Compositions and methods for treatment of pain

ABSTRACT

Embodied herein are engineered fusion proteins that bind and target nociceptor neurons, compositions comprising these engineered fusion proteins, and methods for treatment of pain using these engineered fusion proteins or compositions containing the engineered fusion proteins. The engineered fusion proteins contain domains derived from protein toxins such as the anthrax toxin, clostridial botulinum family of toxins, disulphide-containing toxins, and AB component type toxins.

CROSS REFERENCE TO RELATED APPLICATION

This application claims benefit under 35 U.S.C. § 119(e) of the U.S.provisional application No. 62/210,610 filed Aug. 27, 2015, the contentsof which is/are incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. The ASCII copy, created on Aug. 26, 2016, isnamed 002806-084951-PCT_SL.txt and is 165,499 bytes in size.

FIELD OF THE INVENTION

We describe novel compositions and methods for treatment of pain.

BACKGROUND OF THE INVENTION

Pain in chronic disease conditions including osteoarthritis, rheumatoidarthritis, muscle spasticity, and cancer is a major socioeconomicburden, for which few effective treatments are available.

Current chronic pain therapies such as opioids are mostly ineffective orhave major off-target effects such as addictiveness due to action onother neuronal subtypes.

Nociceptor sensory neurons mediate the detection of harmful/injuriousstimuli, and their aberrant activation produces chronic pain. Theseneurons are dysregulated in muscle spasticity that may contribute tooveractive sensori-motor reflexes, and also innervate joints affected inosteoarticular conditions to mediate pain.

SUMMARY OF THE INVENTION

The present invention provides novel compositions and methods fortreatment of pain. The invention is based, at least in part, on ourdiscovery that pain-sensing nociceptor neurons specifically express highlevels of ANTXR2 (also known as CMG2), a receptor for anthrax toxin,while this receptor is not substantially expressed by other neuronsubtypes. By using the endosomal delivery mechanisms inherent to anthraxtoxin, we can specifically deliver molecular cargo into nociceptors thatwould result in pain-specific block without causing other neurologicalside effects. For examples, the molecular cargoes can be intracellularlyacting toxins that inhibit or block cell signaling pathways in vivo orinhibit or block the release of synaptic neurotransmitters.

Accordingly, we provide, in one aspect, a fusion protein comprising: (a)a botulinum neurotoxin (BTx) or a tetanus neurotoxin (TTx), and (b) ananthrax toxin protective antigen (PA), or a C-terminal receptor-bindingdomain of PA, wherein part (a) and (b) are linked or fused together. Theterm “fusion protein” is used interchangeably with the term “chimericprotein” and the term “engineered fusion protein” herein.

In one embodiment, the BTx or TTx comprises a BTx or TTx enzymaticmoiety and translocation peptide or domain.

In one embodiment, the BTx moiety or translocation peptide/domain isselected from the BTx light chain and heavy chain domains of any one ofBTx/A, BTx/B, BTx/C, BTx/D, BTx/E, BTx/F, BTx/G, and a non-Clostridialbotulinum-like toxin. The back slash followed by an alphabet (/A, /B,/C, etc) denotes the various serotypes within the Clostridium botulinumfamily.

In one embodiment, the BTx or TTx enzymatic moiety or translocationpeptide/domain is selected from the enzymatic moieties and translocationdomain of the BTx or TTx toxins provided in Table 1.

In another aspect, provided herein is a fusion protein comprising: (a) anon-cytotoxic protease, which protease is capable of cleaving a SNAREprotein in a nociceptor neuron; and (b) a protein capable of binding toan anthrax toxin protective antigen (PA) or a fragment thereof, whereinthe PA or PA fragment thereof binds a receptor expressed on thenociceptor neuron. In other words, the fusion protein here cleaves aSNARE protein.

In one embodiment, the non-cytotoxic protease comprises a clostridialneurotoxin L-chain. In one embodiment, the clostridial neurotoxin isbotulinum neurotoxin (BTx) or tetanus neurotoxin (TTx).

In one embodiment, the BTx is selected from any one of BTx/A, BTx/B,BTx/C, BTx/D, BTx/E, BTx/F, BTx/G, and a non-Clostridial botulinum-liketoxin.

In one embodiment, the clostridial neurotoxin L-chain is selected fromthe L-chains of the clostridial neurotoxins provided in Table 1. In oneembodiment, the clostridial neurotoxin L-chain is selected from SEQ. ID.NOS: 20-28.

In another aspect, provided herein is a fusion protein comprising (a) adisulfide-containing peptide toxin which is capable of blocking ionchannels in a nociceptor neuron; and (b) a targeting moiety (TM) that iscapable of binding to a binding site on the nociceptor neuron, whereinthe nociceptor neuron expresses the ion channels therein (e.g., sodiumor calcium or both sodium and calcium channels). In other words, thedisulfide-containing peptide toxin here blocks ion channels in anociceptor neuron and the TM binds to a binding site on the nociceptorneuron.

In another aspect, provided herein is a fusion protein comprising: (a) adisulfide-containing peptide toxin which is capable of blocking sodiumor calcium or both sodium and calcium channels in a nociceptor neuron;and (b) a protein capable of binding to an anthrax toxin protectiveantigen (PA) or a PA fragment that binds a receptor expressed on thenociceptor neuron. In other words, the disulfide-containing peptidetoxin here blocks sodium or calcium or both types of channels in anociceptor neuron and the part (b) is a protein that binds to an anthraxtoxin protective antigen (PA) or a PA fragment that binds a receptorexpressed on the nociceptor neuron.

In one embodiment, the disulfide-containing peptide toxin comprised by afusion protein described herein comprises a cysteine knot motif.

In one embodiment, the disulfide-containing peptide toxin comprised by afusion protein described herein is a conotoxin, an agatoxin, a deltapaulutoxin, a huwentotoxin or a ProTx II toxin.

In one embodiment, the PA-binding receptor expressed on the nociceptorneuron is ANTXR2 (CMG2).

In one embodiment, the PA or C-terminal receptor-binding domain of PAbinds the ANTXR2 (CMG2) receptor expressed on the nociceptor neuron.

In one embodiment, the TM is selected from the group consisting of: (i)an anthrax toxin protective antigen (PA); (ii) a C-terminalreceptor-binding domain of PA; and (iii) a nociceptor neuron-bindingprotein.

In one embodiment, the PA is a mutant PA that is resistant to furincleavage.

In one embodiment, the C-terminal receptor-binding domain of PA is PA63or PAd4.

In one embodiment, the PAd4, the PA or PA fragment thereof, or aC-terminal receptor binding domain of PA that binds ANTXR2 is modifiedor mutated.

In one embodiment, the PAd4, the PA or PA fragment thereof, or aC-terminal receptor binding domain of PA that binds ANTXR2 is resistantto cleavage by a protease, such as Lys C.

In one embodiment, the nociceptor neuron-binding protein is an antibody.

In one embodiment, the antibody specifically binds to the nerve growthfactor (NGF) receptor, the ANTXR2 receptor, or an ion-channel proteinpresent on nociceptor neurons.

In one embodiment, the ion-channel protein is selected from Nav1.7,Nav1.8 or Nav1.9.

In one embodiment, the protein capable of binding to PA is an anthraxtoxin lethal factor (LF) or an anthrax toxin edema factor (EF). In otherwords, the protein here binds PA and is an LF or EF.

In one embodiment, the PA binding domain of LF is the N-terminal domainof LF, (abbreviated as LFPABD or LFn).

In one embodiment, the PA binding domain of EF is the N-terminal domainof EF, (abbreviated as EFPABD or EFn).

In another aspect, provided herein is a fusion protein comprising: (a)an AB toxin; (b) an anthrax toxin protective antigen (PA) or a fragmentthereof, wherein the PA or fragment thereof binds a receptor expressedon a nociceptor neuron; and (c) a translocation domain (TL) that iscapable of translocating the toxin (a protease) from within an endosome,across the endosomal membrane and into the cytosol of the nociceptorneuron. In other words, the TL translocates the toxin into the cytosolof the nociceptor neuron.

In one embodiment, the AB toxin is selected from Ricin toxin, Choleratoxin A-part and B-part; Pseudomonas aeruginosa Exotoxin A A-part andB-part; Shiga toxin A-part and B-part; and Diphtheria toxin A-part andB-part.

In one embodiment, the PA-binding receptor expressed on the nociceptorneuron is ANTXR2 (CMG2).

In one embodiment, the PA fragment is a C-terminal receptor-bindingdomain of PA.

In one embodiment, the TL is a translocation domain derived from aclostridial neurotoxin, or is a holotoxin; or is a mutant form of theholotoxin that has been modified (e.g., chemically) or mutated to negatethe toxin receptor-binding function of the AB toxin.

In another aspect, provided herein is a fusion protein comprising abotulinum neurotoxin (BTx) moiety comprising an N-terminal enzymaticdomain (LC or L chain) and an intermediatepore-forming/translocation-domain (H_(N)) of the BTx, linked to aC-terminal receptor-binding domain of anthrax toxin protective antigen(PA). The C-terminal receptor-binding domain of anthrax toxin protectiveantigen can be, for example, a PAd4 domain.

In one embodiment, the fusion protein further comprises a linker peptidebetween the BTx moiety and the C-terminal receptor-binding domain ofanthrax toxin protective antigen or PAd4 domain.

In another aspect, provided herein is a fusion protein comprising: (a) abotulinum neurotoxin N-terminal enzymatic domain of a botulinumneurotoxin (BTx) moiety, and (b) an N-terminal domain of anthrax toxinlethal factor (LFn), which domain binds to oligomeric forms of PA63, theproteolytically activated form of anthrax PA; or the N-terminal domainof anthrax toxin edema factor (EFn), which domain binds to oligomericforms of PA63, the proteolytically activated form of anthrax PA, whereinpart (a) is linked N-terminally or C-terminally or both N-terminally andC-terminally to part (b).

In one embodiment of any of the fusion proteins including a BTx moiety,the fusion protein can further comprise an amino acid sequence defininga belt corresponding to the N-terminal part of the BTx H_(N) domain,wherein the H_(N) of the BTx is located at the C-terminal side of theBTx moiety. The presence of the belt stabilizes the L chain.

In one embodiment of any of the fusion proteins including a BTx moiety,wherein the BTx moiety comprises, consist essentially of, or consists ofthe L chain and the H_(N) domain of BTx, the S-S bridge between the Lchain and the H_(N) domain is not reduced.

In one embodiment of any of the fusion proteins including a BTx moiety,wherein the BTx moiety comprises, consist essentially of, or consists ofthe L chain and not the H_(N) domain of BTx, the Cys residues in the Lchain and the belt corresponding to the N-terminal part of the BTx H_(N)domain, if present, can be mutated to Ala, Ser, or Thr.

In one embodiment, for a fusion protein comprising a BTx L moiety and anLFn or EFn domain, the fusion protein further comprises a linker peptidebetween the BTx L moiety and the LFn or EFn domain.

In another aspect, provided herein is a fusion protein comprisinganthrax toxin protective antigen (PA), an anthrax toxin protectiveantigen C-terminal receptor binding domain (PAd4), or a nociceptorneuron-binding protein, linked to a disulfide-containing peptide toxin.In one embodiment, the disulfide-containing peptide toxin is aninhibitor cysteine knot toxin.

In one embodiment, the fusion protein further comprises a linker peptidebetween the PA, PAd4 or nociceptor-binding protein and thedisulfide-containing peptide toxin (e.g., the inhibitor cysteine knottoxin).

In another aspect, provided herein is a fusion protein comprising adisulfide-containing peptide toxin operably linked N-terminally orC-terminally or both N-terminally and C-terminally, or chemicallycrosslinked at one or more sites, to the N-terminal domain (LFn) ofanthrax toxin lethal factor, which domain binds to oligomeric forms ofPA63, the proteolytically activated form of anthrax PA; or theN-terminal domain (EFn) of anthrax toxin edema factor, which domainbinds to oligomeric forms of PA63.

In one embodiment of any fusion protein described in which a toxin isfused to LFn or EFn, the fusion protein further comprises a linkerpeptide between the LFn and the toxin or the EFn and the toxin.

In another aspect, provided herein is a fusion protein comprising an ABtoxin fused to a linker peptide linked to a C-terminal receptor-bindingdomain of anthrax toxin protective antigen (PAd4 domain), wherein thefusion protein further comprises a translocation domain, a holotoxin, ora mutant form of the holotoxin that have been modified (e.g.,chemically) or mutated to negate the toxin receptor-binding function ofthe AB toxin.

In one embodiment of any fusion protein including an AB toxin, the ABtoxin is selected from Ricin toxin, Cholera toxin A-part and B-part,Pseudomonas aeruginosa Exotoxin A A-part and B-part, Shiga toxin A-partand B-part, and Diphtheria toxin A-part and B-part.

In another aspect, provided herein is a fusion protein comprising anN-terminal enzymatic domain (Chain A) together with atranslocation/pore-forming domain from a Clostridial neurotoxin or anon-Clostridial botulinum-like toxin, linked to a C-terminalreceptor-binding domain of anthrax toxin protective antigen (PAd4domain). Examples of a Clostridial neurotoxin is tetanus neurotoxin(frequently abbreviated as TTx or TeNT in scientific literatures).

In one embodiment, the fusion protein further comprises a linker peptidebetween the TTx moiety and the PAd4 domain.

In one embodiment of any fusion protein described as having a linker,the linker peptide is 1-20 amino acids long.

In one embodiment of any fusion protein described as having a linker,the linker peptide is stable in human serum for at least 1 minute.

In one embodiment of any fusion protein described as having a linker,the linker peptide comprises at least one amino amino acid that is Glyor Ser.

In one embodiment of any fusion protein described as having a linker,the linker peptide does not comprise Lys and/or Arg.

In one embodiment of any fusion protein described that includes a BTxmoiety, the BTx moiety is selected from the BTx light chain and heavychain domains of any one of BTx/A, BTx/B, BTx/C, BTx/D, BTx/E, BTx/F,BTx/G. For examples, the the BTx light chain and heavy chain domains areselected from SEQ ID NO: 29-31 or Table 1 described herein. It isspecifically contemplated that a non-Clostridial botulinum-like toxincan also be used.

In one embodiment of any fusion protein described as including a PAd4domain, the fusion protein comprises 2-10 PAd4 domains in tandem.

In one embodiment of any fusion protein described as including a PAd4domain and a BTx moiety, about 1-60 consecutive amino acids from theN-terminal side of PA adjacent to the native PAd4 domain are furtherincorporated between the BTx moiety and the PAd4.

In one embodiment of any fusion protein described as including a PAd4domain and an AB toxin, about 1-60 consecutive amino acids from theN-terminal side of PA adjacent to the native PAd4 domain are furtherincorporated between the AB toxin and the PAd4.

In one embodiment of any fusion protein described as including a PAd4domain, e.g., a PAd4, a PA or a C-terminal receptor binding domain ofPA, one or more of the Lys residues in the PAd4 domain at positions 594,613, 633, 637, 653, 673, 679, 680, 684, 695, 703, 722, 723, 729, and 730has been replaced by Arg or His, wherein the numbering refers to that ofSEQ ID NO:1 after minusing the 29 aa signal peptide in SEQ. ID. NO:1.

In one embodiment of any fusion protein described as including a PAd4domain, e.g., a PA or a C-terminal receptor binding domain of PA, one ormore of the Lys residues in the PAd4 domain at positions 623, 642, 662,666, 682, 702, 708, 709, 713, 724, 732, 751, 752, 758, and 759 in SEQ.ID. NO:1 has been replaced, for example, by Arg or His.

In one embodiment of any fusion protein described as including an entirePA protein, the furin cleavage site of PA comprising amino acid residues¹⁶⁴RKKR¹⁶⁷ of SEQ ID NO: 1 (minus the 29 aa signal peptide in SEQ. ID.NO:1) has been replaced by a furin-resistant amino acid sequence. In oneembodiment, the furin-resistant amino acid sequence is SSSR (SEQ ID NO:32) or SSSS (SEQ ID NO: 33).

In one embodiment of any fusion protein described as including a PAd4domain, one or more of the Asn residues in the PAd4 domain at position601, 713, 719 of SEQ ID NO:1 (minus the 29 aa signal peptide in SEQ. ID.NO:1) has been replaced by Asp.

In one embodiment of any fusion protein described herein, the fusionprotein further comprises at least one D-amino acid at the N-terminus ofthe fusion protein.

In one embodiment of any fusion protein that includes all or part of aBTx L chain and H chain, the residue corresponding to an L chainjunction of BTx with an H chain of BTx has been cleaved.

In one embodiment of any fusion protein that includes a nociceptorneuron-binding protein, the nociceptor neuron-binding protein is anantibody. In one embodiment, the antibody specifically binds to NGFreceptor or an ion-channel protein present on nociceptor neurons. In oneembodiment, the ion-channel protein is selected from Nav1.7, Nav1.8 orNav1.9.

In another aspect, provided herein is a composition comprising any oneof the fusion proteins described in the preceding paragraphs. In oneembodiment, the composition further comprises a pharmaceuticallyacceptable carrier, excipient or diluent.

In one embodiment of any composition described, the composition furthercomprises a native anthrax toxin protective antigen (PA). In oneembodiment, the PA is an oligomeric PA. In one embodiment, theoligomeric PA is bound to the fusion protein.

In another aspect, provided herein is a nucleic acid encoding any of thefusion proteins described herein.

In another aspect, provided herein is a vector comprising the nucleicacid described in the preceding paragraph. For examples, the vector is aplasmid, a bacteriophage or a viral vector. For example, the plasmid isan expression plasmid for recombinant protein expression in a bacteria,e.g., Escherichia coli. In another aspect, provided herein is a viralparticle comprising a vector comprising a nucleic acid described in thepreceding paragraph. In another aspect, provided herein is a viralparticle comprising a a nucleic acid described in the precedingparagraph.

In another aspect, provided herein is a cell comprising the nucleic aciddescribed herein or the vector of described herein. For example, an E.coli carrying a plasmid that comprises a nucleic acid encoding a fusionprotein described herein. For example, for the recombinant proteinexpression of the a fusion protein encoded in the nucleic acid. Inanother aspect, provided herein is a cell comprising a viral particlecomprising a vector comprising a nucleic acid described in the precedingparagraph. In another aspect, provided herein is a cell comprising aviral particle comprising a a nucleic acid described in the precedingparagraph.

In another aspect, provided herein is a method of producing a fusionprotein, the method comprising culturing the cell described herein aboveunder conditions such that the fusion protein is expressed, andrecovering the fusion protein.

In another aspect, provided herein is a fusion protein produced by themethod described in the preceding paragraph.

In one embodiment, any of the fusion proteins described herein, or afusion protein produced by a method described herein, is glycosylated.In another embodiment, any of the fusion proteins described herein, or afusion protein produced by a method described herein, isnon-glycosylated.

In one embodiment of the method described for producing a fusionprotein, the cell is a prokaryotic cell such as bacteria. In oneembodiment of the method described for producing a fusion protein, thecell is a bacteria cell. In one embodiment, the bacteria is Escherichiacoli (E. coli). In another embodiment, the bacteria is an attenuatedBacillus anthracis strains, e.g., CDC 684. In one embodiment of themethod described for producing a fusion protein, the cell is a yeastcell. In one embodiment, the yeast is Saccharomyces cerevisiae.

In one embodiment of the method described for producing a fusionprotein, the yeast cell is glycosylation deficient.

In one embodiment of the method described for producing a fusionprotein, the yeast cell is glycosylation and protease deficient.

In one embodiment of the method described for producing a fusionprotein, the cell is a mammalian cell. In one embodiment, the mammaliancell is a COS cell, a CHO cell, or an NSO cell.

In one embodiment, provided herein is the use of any of the fusionproteins described herein for the treatment of pain.

In one embodiment, provided herein is the use of any of the fusionproteins described herein for the manufacture of medicament for thetreatment of pain.

In another aspect, provided herein is an engineered fusion proteincomprising an anthrax toxin Protective-Antigen (PA) moiety or itsreceptor binding domain (PAd4) fused with an inhibitor cysteine knot(ICK) toxin, e.g., a Conotoxin (CTx).

In another aspect, provided herein is an engineered fusion proteincomprising an anthrax toxin lethal factor domain (LFn) fused with aninhibitor cysteine knot (ICK) toxin and a Protective-Antigen (PA)moiety. In another aspect, provided herein is an engineered fusionprotein comprising an anthrax toxin lethal factor domain (LFn) fusedwith a L chain of a Clostridial neurotoxin and a Protective-Antigen (PA)moiety. In one embodiment, this fusion protein can further comprise thebelt of the H chain of the Clostridial neurotoxin, the belt is theN-terminal segment of the H chain. In another aspect, provided herein isan engineered fusion protein comprising an anthrax toxin lethal factordomain (LFn) fused with an intracellular acting toxin (e.g., an AB typetoxin) and a Protective-Antigen (PA) moiety.

In another aspect, provided herein is an engineered fusion proteincomprising a mutant anthrax protective antigen (mPA) moiety that hasbeen altered to block its native receptor-binding function, fused with amolecule capable of specifically targeting a nociceptor surface receptoror a nociceptor ion channel receptor, and an anthrax lethal factordomain (LFn) fused to an intracellularly acting toxin catalytic domain.ANTXR2 is the native receptor for PA. In other words, the moleculespecifically targets a nociceptor surface receptor or a nociceptor ionchannel receptor.

In another aspect, provided herein is an engineered fusion proteincomprising an anthrax protective antigen (PA) moiety fused with amolecule capable of specifically targeting a nociceptor surface receptoror a nociceptor ion channel receptor, and an anthrax lethal factordomain (LFn) fused to an intracellularly acting toxin catalytic domain.ANTXR2 is the native receptor for PA. In some embodiments of any of theaspects described herein, PA can be further engineered to enhancebinding to one or more receptors. In other words, the moleculespecifically targets a nociceptor surface receptor or a nociceptor ionchannel receptor.

In one embodiment, the molecule capable of specifically targeting or themolecule that specifically targets a nociceptor surface receptor or anociceptor ion channel receptor is selected from an antibody, orantibody mimetic, that specifically binds to the NGF receptor, or anantibody or antibody mimetic that specifically binds to Nav1.7, Nav1.8or Nav1.9. In some embodiments of any of the aspects described herein,molecule capable of specifically targeting a nociceptor surface receptoror a nociceptor ion channel can be further engineered to enhance bindingto one or more receptors.

In one embodiment, wherein the intracellularly acting toxin catalyticdomain is selected from diphtheria toxin (DTx), Pseudomonas aeruginosaexotoxin A (PTx), botulinum toxin (BTx), tetanus toxin (TTx), shigatoxin, ricin toxin, anthrax lethal toxin (lethal factor, LF), and/oranthrax edema toxin (edema factor, EF).

In another aspect, provided herein is an engineered fusion proteincomprising a native protective antigen (PA) or a mutant PA (mPA),wherein the mPA has been modified (e.g., chemically) or mutated so as toblock its native receptor-binding function, and a molecule that cantarget nociceptor neuron surface molecules, specifically in combinationwith anthrax toxin edema factor (EF) and/or anthrax lethal factor (LF).

In one embodiment of an engineered fusion protein of any of thepreceding paragraphs, the PA or mPA is in a covalent or noncovalentoligomeric form. In one embodiment, the oligomeric form is bound to themolecule, e.g., covalently or noncovalently.

In another aspect, provided herein is a composition comprising anengineered fusion protein comprising an anthrax toxin Protective-Antigen(PA) moiety or its receptor binding domain (PAd4) fused with aninhibitor cysteine knot (ICK) toxin, e.g. a Conotoxin (CTx).

In another aspect, provided herein is a composition comprising anengineered fusion protein comprising an anthrax toxin lethal factordomain (LFn), fused with an inhibitor cysteine knot (ICK) toxin (e.g., aconotoxin (CTx)) and a Protective-Antigen (PA) moiety.

In another aspect, provided herein is a composition comprising anengineered mutant anthrax protective antigen (mPA) moiety that has beenaltered to block its native receptor-binding function, fused with amolecule capable of specifically targeting a nociceptor surfacereceptor, or a nociceptor ion channel and an anthrax lethal factordomain (LFn), fused to an intracellularly acting toxin catalytic domain.

In another aspect, provided herein is a composition comprising anengineered fusion protein comprising an anthrax protective antigen (PA)moiety fused with a molecule capable of specifically targeting anociceptor surface receptor or a nociceptor ion channel receptor, and ananthrax lethal factor domain (LFn) fused to an intracellularly actingtoxin catalytic domain. ANTXR2 is the native receptor for PA. In someembodiments of any of the aspects described herein, PA can be furtherengineered to enhance binding to one or more receptors.

In one embodiment, the molecule capable of specifically targeting anociceptor surface receptor or a nociceptor ion channel is selected froma NGF, and an antibody that specifically binds to Nav1.7, Nav1.8 orNav1.9. In some embodiments of any of the aspects described herein,molecule capable of specifically targeting a nociceptor surface receptoror a nociceptor ion channel can be further engineered to enhance bindingto one or more receptors.

In another embodiment, the intracellularly acting toxin catalytic domainis selected from diphtheria toxin (DTx), Pseudomonas aeruginosa exotoxinA (PTx), botulinum toxin (BTx) tetanus toxin (TTx), Shiga toxin, ricintoxin, anthrax lethal toxin (lethal factor, LF), and/or anthrax edematoxin (edema factor, EF).

In another aspect, provided herein is a composition comprising anengineered fusion protein comprising a native protective antigen (PA) ora mutant PA (mPA), wherein the mPA has been modified (e.g., chemically)or mutated so as to block its native receptor-binding function, and amolecule that can target nociceptor neuron surface moleculesspecifically in combination with anthrax toxin edema factor (EF).

In one embodiment, the PA or mPA is in an oligomeric form. In oneembodiment, the oligomeric form is bound to the molecule.

In one embodiment, a fusion protein-comprising composition furthercomprises a pharmaceutically acceptable carrier or excipient.

In another aspect, provided herein is a method for treatment of pain,the method comprising administering to a subject in need thereof acomposition comprising a fusion protein as described herein.

In another aspect, provided herein is a method of treating pain, themethod comprising administering to a subject in need thereof a nativemature anthrax toxin protective antigen (PA) and anthrax toxin edemafactor (EF), anthrax toxin lethal factor (LF) or any combinationthereof.

In another aspect, provided herein is a method for the treatment ofnerve, joint, skin, visceral, bladder, or muscle pain, comprisingadministering peripherally by intradermal injection, subcutaneousinjection, intramuscular injection, intraneural injection, orintra-articular injection to a subject in need thereof a compositioncomprising a fusion protein as described herein.

In another aspect, described herein is a method for treatment ofdiabetic neuropathic pain, cancer pain, fibromyalgia or other systemicpain disorders, comprising administering by epidural injection,intrathecal infusion or intra-cerebroventricular infusion into thecentral nervous system of a subject in need thereof a compositioncomprising a fusion protein as described herein.

In another aspect, described herein is a method for treatment of pain,the method comprising administering to a subject in need thereof aneffective, pain reducing amount of a composition comprising anengineered fusion protein comprising an anthrax toxin Protective-Antigen(PA) moiety or its receptor binding domain (PAd4) fused to anintracellularly acting toxin catalytic domain, wherein the engineeredfusion protein is delivered to nociceptor neurons and results indecreased intracellular signaling events in the nociceptor neurons ordecreased neurotransmitter release from the nociceptor neurons.

In one embodiment, the intracellularly acting toxin catalytic domain isselected from diphtheria toxin (DTx), Pseudomonas aeruginosa exotoxin A(PTx), botulinum toxin (BTx) tetanus toxin (TTx), Shiga toxin, ricintoxin, anthrax lethal toxin (lethal factor), and/or anthrax edema toxin(edema factor)

In some aspects, any compositions described in the preceding paragraphsor any compositions comprising a fusion protein described in thepreceding paragraphs is use for the treatment of pain. Treatment of paincan include administering more than one, i.e., several, of the differentcompositions described in the preceding paragraphs.

In another aspect, described herein is a method for treatment of pain,the method comprising administering to a subject in need thereof aneffective, pain reducing amount of a composition comprising anengineered fusion protein comprising an anthrax toxin Protective-Antigen(PA) moiety or its receptor binding domain (Pad4) fused with aninhibitor cysteine knot (ICK) toxin (e.g., a Conotoxin (CTx)).

In another aspect, described herein is a method for treatment of pain,the method comprising administering to a subject in need thereof aneffective amount of a composition comprising an engineered fusionprotein comprising an anthrax toxin lethal factor (LFn) fused with aninhibitor cysteine knot (ICK) toxin (e.g., a Conotoxin (CTx)) and aProtective-Antigen (PA) moiety. Alternately, treatment of pain iscarried out by administering to a subject in need thereof an effectiveamount of a composition comprising an engineered fusion proteincomprising an anthrax toxin lethal factor domain (LFn) fused with a Lchain of a Clostridial neurotoxin and a Protective-Antigen (PA) moiety,or an engineered fusion protein comprising an anthrax toxin lethalfactor domain (LFn) fused with an intracellular acting toxin (e.g., anAB type toxin) and a Protective-Antigen (PA) moiety. In one embodiment,the fusion protein comprising the L chain of a Clostridial neurotoxincan further comprise the belt of the H chain of the Clostridialneurotoxin, the belt is the N-terminal segment of the H chain.

In another aspect, described herein is a method for treatment of pain,the method comprising administering to a subject in need thereof aneffective, pain reducing amount of an engineered mutant anthraxprotective antigen (mPA) moiety that has been altered to block itsnative receptor-binding function fused with a molecule capable ofspecifically targeting a nociceptor surface receptor or an ion channelreceptor and an anthrax lethal factor domain (LFn) fused to anintracellularly acting toxin catalytic domain.

In one embodiment, the molecule is selected from an antibody thatspecifically binds to the NGF receptor and an antibody that specificallybinds to Nav1.7, Nav1.8 or Nav1.9.

In another embodiment, the intracellularly acting toxin catalytic domainis selected from diphtheria toxin (DTx), Pseudomonas aeruginosa exotoxinA (PTx), botulinum toxin (BTx), tetanus toxin (TTx), Shiga toxin, ricintoxin, anthrax lethal toxin (lethal factor), and/or anthrax edema toxin(edema factor).

In another aspect, described herein is a method of treating pain in asubject in need thereof comprising administering to the subject anengineered fusion protein comprising a native protective antigen (PA) ora mutant PA (mPA), wherein the mPA has been modified (e.g., chemically)or mutated so as to block its native receptor-binding function and amolecule that can target nociceptor surface molecules specifically incombination with anthrax toxin edema factor (EF) and/or anthrax lethalfactor (LF).

In one embodiment, the PA or mPA is administered in an oligomeric form,wherein the oligomeric PA or mPA is formed from proteolyticallyactivated PA or mPA (or mutant thereof) to achieve increased avidity forreceptor-bearing cells. In one embodiment, the oligomeric form is boundto the molecule before administering.

In one embodiment, the composition is administered separately before,simultaneously, or after administering a composition comprising ananthrax protective antigen (PA), in a pharmaceutically acceptablecarrier, excipient or diluent.

In one embodiment, the administering is performed by intrathecalinfusion or intra-cerebroventricular infusion or by an epiduralinjection into the central nervous system, or by peripheraladministration using intradermal injection, subcutaneous injection,intramuscular injection, intraneural injection, or intra-articularinjection.

In another embodiment, the pain is selected from diabetic neuropathicpain, cancer pain, fibromyalgia, other systemic pain disorders, nerve,joint, skin, visceral, bladder, and muscle pain.

In another aspect, provided herein is a method of manufacture of apharmaceutical composition comprising one or more of the fusion proteinsdescribed in the preceding paragraphs and a pharmaceutically acceptablecarrier or excipient.

In another aspect, provided herein is a fusion protein described in thepreceding paragraphs for use in the manufacture of medicament for thetreatment of pain. In one embodiment, the fusion protein is formulatedwith at least one pharmaceutically acceptable carrier or excipient.

In another aspect, provided herein is a fusion protein described in thepreceding paragraphs for use in the treatment of pain. In oneembodiment, the fusion protein is formulated with at least onepharmaceutically acceptable carrier or excipient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B demonstrate that Anthrax Toxin Receptor is specificallyexpressed in the dorsal root ganglia compared to other nervous systemtissues. (FIG. 1A) Expression profiling data of 11 nervous tissue typesshowing that Antxr2 transcript expression is only present in dorsal rootganglia tissues, where nociceptor neurons are found. (FIG. 1B) In situhybridization image for Antxr2 showing strong expression in dorsal rootganglia but not surrounding tissues or spinal cord.

FIG. 2 demonstrates that Antxr2 is highly enriched in nociceptor neuronscompared to proprioceptor sensory neurons (large dot and arrow). Volcanoplot (P-value vs. fold-change of difference) shows Antxr2 is stronglyenriched in nociceptor pain-sensing neurons compared to proprioceptorneurons.

FIG. 3 shows that the Protective Antigen (PA) alone does not inhibitprotein synthesis in neurons.

FIG. 4 shows that PA alone does not inhibit protein synthesis inneurons.

FIG. 5 shows that protein synthesis inhibition in neurons by the fusionprotein LFn-DTX is dependent on the presence of PA and that LFn-DTX isable to block protein synthesis intracellularly in nociceptor neurons atpicomolar concentrations.

FIG. 6 shows that PA and the fusion protein LFn-DTX inhibit proteinsynthesis in neurons.

FIG. 7A shows the modular construction of embodiments of BoTX-PA fusionproteins using various domains of BoTX protein and different PA-derivedproteins. The PA-derived protein is the Pad4 domain or PA that isresistant to cleavage by a protease, such as furin protease.

FIG. 7B shows the modular construction of embodiments of TTX-PA fusionproteins using various domains of TTX protein and different PA-derivedproteins. The PA-derived protein is the Pad4 domain or PA that isresistant to cleavage by a protease, such as furin protease.

FIG. 8A shows the modular construction of embodiments of a fusionproteins comprising BoTX and the PA-binding domains LFn or EFn. Thefusion proteins are made using the light chain/catalytic domain fromvarious BoTX serotypes with the PA-binding domain of the two PA-bindingproteins, LF and EF. These BoTX-PA-binding fusion proteins are to beused in conjunction with the native PA protein for the treatment ofpain.

FIG. 8B shows the modular construction of embodiments of a fusionproteins comprising TTX or other intracellular acting toxins and thePA-binding domains LFn or EFn. These fusion proteins are made using thelight chain/catalytic domain of TTX protein or various otherintracellular acting toxins, with the PA-binding domain of the twoPA-binding proteins, LF and EF.

FIG. 9 shows the modular construction of embodiments of a fusionproteins comprising small disulfide containing toxins or inhibitorcysteine knots (ICK) toxins and a PA-derived protein. The PA-derivedprotein is the Pad4 domain or the PA that is resistant to by a protease,such as furin protease.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations: ANTXR2 (CMG2)=a cell surface receptor for the anthraxtoxin; PA=anthrax toxin Protective-Antigen, 83 kDa; PA63=active 63 kDaof fragment of PA derived from furin cleavage, self-assembles into aring-shaped heptamer or octamer to form a receptor-bound prepore, PA63prepore binds up to three or four EF, LF, LFn or EFn, forming complexesthat are then endocytosed; PAd1=an LF/EF binding component or fragmentof PA; PAd2=a membrane translocation component or fragment of PA, ananthrax-derived translocation domain or peptide; PAd3=an oligomerisationcomponent or fragment of PA; PAd4=the host cell receptor binding domainof PA to ANTXR1 and ANTXR2 receptors, PA's native receptors;PA^(furin-)=a furin resistant PA with modified or mutated furin-proteaserecognition site, is incapable of multimerisation and translocation, nobinding to LFn or EFn but can still bind host cell receptor;ICK=inhibitor cysteine knot; LFn=N-terminal PA binding domain of anthraxtoxin lethal factor, an “anthrax toxin translocation peptide”;EFn=N-terminal PA binding domain of anthrax toxin edema factor, also an“anthrax translocation signal peptide”; LF=anthrax lethal toxin (lethalfactor); EF=anthrax edema toxin (edema factor); mPA=a mutant anthraxprotective antigen moiety that has been altered to block its nativereceptor-binding function; Nav1.7, Nav1.8 or Nav1.9=ion channelproteins; DTx=diphtheria toxin including A and B components of thetoxin, DTA=diphtheria toxin only the A components of the toxin, theenzymatic component, the component that is the intracellular actingtoxin; PE or PTx=Pseudomonas aeruginosa exotoxin A; BTx or BoTX orBoNT=botulinum toxin; TTx=tetanus toxin; CTx=Conotoxin; CNT=clostridialneurotoxin family; LC or L=50 kDa light chain of of a neurotoxin memberof the clostridial neurotoxin family, L functions as a zinc-dependentendopeptidase; HC=heavy chain (HC=H_(N)+H_(C)) contains two functionaldomains, each of ˜50 kDa; H_(N)=N-terminal half of HC, is thetranslocation domain of a neurotoxin member of the clostridialneurotoxin family. H_(N) is known to form ion channels in lipidbilayers. H_(C)=C-terminal half of HC, is the receptor binding domain ofa neurotoxin member of the clostridial neurotoxin family;LH_(N)=L+H_(N).

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art to which thisdisclosure belongs. It should be understood that this invention is notlimited to the particular methodology, protocols, and reagents, etc.,described herein and as such can vary. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims. Definitions of common terms in molecular biologycan be found in The Merck Manual of Diagnosis and Therapy, 19th Edition,published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular CellBiology and Molecular Medicine, published by Blackwell Science Ltd.,1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), MolecularBiology and Biotechnology: a Comprehensive Desk Reference, published byVCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by WernerLuttmann, published by Elsevier, 2006; Janeway's Immunobiology, KennethMurphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014(ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones &Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green andJoseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012)(ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology,Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.)Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology(CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN047150338X, 9780471503385), Current Protocols in Protein Science (CPPS),John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and CurrentProtocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David HMargulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons,Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which areall incorporated by reference herein in their entireties.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

We have identified a novel way to treat pain utilizing pain-sensingnociceptor-specific delivery of molecules that quell pain.

Skin lesions caused by Bacillus anthracis, the causative agent ofanthrax, are characteristically painless. We discovered thatpain-sensing nociceptor neurons specifically express high levels ofANTXR2, the receptor for anthrax toxin, while this receptor is notexpressed or substantially expressed by other neuron subtypes. Anthraxtoxin receptor is also expressed by hematopoietic lineage cells(macrophages, osteoclasts, osteoblasts) involved in joint remodeling.Without wishing to be bound by a theory, we submit that anthrax toxinsilences pain during infection by acting through ANTXR2 on nociceptorsensory neurons, and that it can also be used to target immune cellsinvolved in joint remodeling.

We have previously described a system of targeted delivery of antigens(U.S. Patent Application Publication No. 20030202989) and proteins(WO2012096926) using different anthrax toxin-based delivery systems,including a system in which anthrax receptor binding to its nativetarget receptor has been ablated (U.S. Patent Application PublicationNo. 20150044210). In these systems, the pore-forming ability of anthraxtoxin was exploited to permit entry of a reagent into a cell. Thereceptor-binding portion of anthrax toxin was ablated in order to permitengineering of cell-binding specificity as desired by the user.

In short, the receptor-binding component of anthrax toxin, termedProtective Antigen (PA), binds to ANTXR2, is endocytosed, andsubsequently translocates either anthrax Lethal Factor (LF) or anthraxEdema Factor (EF), or both, across the endosomal membrane to thecytosol. As described herein, the inventors have made the surprisingdiscovery that the major anthrax toxin receptor ANTXR2 is highlyspecific in expression within nociceptive neurons amongst all nervoustissues, i.e., anthrax toxin is preferentially targeted to nociceptiveneurons via binding to ANTXR2. The discovery of this preferentialbinding of the native anthrax toxin permits the use of anthrax toxin andits cytosolic delivery mechanisms to specifically target nociceptiveneurons to produce highly specific and efficacious pain blockade. Insome embodiments of any of the aspects described herein, no receptorbinding modifications are needed, as the target neurons express thereceptor the anthrax system naturally binds to. In some aspects of anyof the embodiments, specificity can be increased and side effectsreduced by use of additional ligand-receptor interactions, in concertwith the anthrax toxin-ANTXR2 interaction. Here we utilized anthraxtoxin as a platform to specifically block chronic pain, silence musclespasticity, and to target and prevent both pain and joint destruction inosteoarthritis. The methods and compositions described herein divergefrom earlier anthrax-toxin delivery systems in utilizing thereceptor-binding portion of anthrax toxin to direct delivery to aspecific cell type, instead of using only the pore-forming activity ofthe toxin to permit entry of a reagent to a cell targeted by meansindependent of any native anthrax toxin binding activity.

Chronic pain is a major socio-economic burden in society for which fewtargeted treatments are available. Nociceptor sensory neurons mediatethe detection of noxious/injurious stimuli, resulting in pain sensationsand avoidance behavior. Sustained activation of nociceptors duringinflammation or following nerve injury leads to chronic pain. We provideuses of proteinaceous toxins to create new targeted molecular entitiesto treat pain.

We have found that the anthrax toxin receptor ANTXR2 is highly expressedon nociceptors and specific to these neurons compared to other neuronalsubtypes. This finding suggests that we can use the Protective Antigen(PA) moiety of anthrax toxin or its receptor-binding domain (PAd4) as acellular specificity determinant for creating constructs to killnociceptors or otherwise block their ability to transmit signals to thecentral nervous system (CNS).

The advantage of this surprising strategy is the specificity oftargeting peripheral pain-sensing neurons compared to other paintreatments, which often have off-target effects on other neuronalsubtypes.

We describe engineering intracellularly acting toxins such as anthraxtoxin, disulfide-containing peptide toxins such as inhibitor cysteineknot (ICK) toxins, AB type toxins such as diphtheria toxin (DT), andSNARE targeting toxins such as tetanus toxin (TTx), and/or botulinumtoxins (BTx) for use as a targeted painkiller. In some embodiments,these toxins depend on PA mediated delivery of the intracellularenzymatic activity of these toxins into nociceptor neurons.

Toxins and their Components, Parts and Fragments Useful for the ModularConstruction of Engineered Fusion (Chimeric) Proteins

Anthrax Toxin

Anthrax toxin is a trimeric complex of three protein components secretedby virulent strains of the bacterium, Bacillus anthracis. The threeprotein components are Protective Antigen (PA), Edema Factor (EF), andLethal Factor (LF). PA is an 83 kDa protein that mediates specificreceptor targeting and binding. Upon binding to its receptor, eitherTumor Endothelium Marker-8 (TEM8 or “ANTRX1”) or Capillary MorphogenesisProtein 2 (CMG2 or “ANTXR2”), PA83 is proteolytically activated by furinor other furin-like proteases, removing an N-terminal piece (PA20) andleaving the remaining piece (PA63) bound to the receptor. This activatesPA enabling it to multimerise to form heptamers/octamers that bind up to4 EF/LF molecules. PA63 spontaneously oligomerizes to form ring-shapedheptameric or octameric “prepores”, which contain binding sites capableof binding LF or EF with high nM affinity. LF and EF (each ˜90 kDa) havehomologous, ˜260-residue N-terminal domains that bind the prepores; theenzymatic moieties of LF and EF are C-terminal. The resulting complexesare internalised by endocytosed and under low pH of the endosome, thePA63 prepore changes conformation, inserts into the membrane andtransports bound cargo molecules (LF/EF) to the cytosol, where theyrefold and catalyze their respective reactions. The LF is zinc dependentendopeptidase that catalyzes the hydrolysis (cleavage) of certainmitogen-activated protein kinase kinases (MAP Kinase/ERK Kinase; alsoknown as MAP Kinase Kinase), and this leads to the disruption of manycellular signalling pathways, which eventually leads to cell death. EFis a calmodulin and calcium dependent adenylate cyclase that increasescAMP to extraordinary levels in cells. Changes in intracellular cAMPaffect membrane permeability and may account for edema. In macrophagesand neutrophils, an additional effect is the depletion of ATP reserveswhich are needed for the engulfment process. This is a list of thepossible combinations that can occur with the Anthrax Toxin: PA+LF leadsto lethal activity; EF+PA leads to edema; EF+LF has no toxic effects oncells; and PA+LF+EF leads to lethal activity and edema in an affectedcell. LF consists of an N-terminal PA binding domain (abbreviated hereinas “LFPABD” or “LFn”) and a C-terminal proteolytic component. LF acts bycleaving mitogen-activated protein (MAP) kinase kinases. EF consists ofan N-terminal PA binding domain (abbreviated herein as “EFPABD” or“EFn”), a central enzymatic component, and a C-terminal calmodulinbinding component. EF is a calcium dependent adenylate cyclase thatelevates cellular cAMP levels. MAP kinase and cAMP signalling have bothbeen found to be critical in mediating nociceptor signalling.

PA possesses four major functions distributed across four structurallydistinct domains—an LF/EF binding component (PAd1), a membranetranslocation component (PAd2), an oligomerisation component (PAd3), anda host cell receptor binding component (PAd4).

As used herein, “anthrax toxin protective antigen” or “PA” refers to apolypeptide that, in oligomeric form, binds specifically and selectivelyto the ANTXR2 receptors, subsequently forming a pore in the cellmembrane and translocating cargo toxins. The sequence of PA is known inthe art, e.g., (NCBI Gene ID No: 3361714 (SEQ ID NO: 1; the 29 aminoacid residue peptide sequence is the signal peptide:MKKRKVLIPLMALSTILVSSTGNLEVIQA (SEQ ID NO: 34) at the N-terminus (NCBIRef Seq: NP_052806; UNIPROT P13423). The numbering of the amino acidresidues can be with reference to SEQ. ID. NO: 1 that has the signalpeptide. Alternatively, the numbering of the amino acid residues can bewith reference to the PA sequence without the signal peptide. PA bindsto host cell surface ANTXR2 receptors and is cleaved by a furin-familyprotease to an active 63 kDa PA form (PA63) that self-assembles into aring-shaped heptamer or octamer to form a receptor-bound prepore. ThePA63 prepore binds up to three or four molecules of, e.g., anthraxlethal factor, forming complexes that are then endocytosed. Uponacidification of the endosome, protective antigen prepore undergoes aconformational rearrangement to form a membrane-spanning, ion-conductivepore, which transports anthrax lethal factor and/or anthrax edema factorfrom the endosome to the cytosol. LFn, the N-terminal domain of anthraxlethal factor, has nanomolar binding affinity for the pore, and thisdomain (or the corresponding EF domain, EFn) alone can be used fortranslocation of chemical moieties.

The furin-family protease cleavage site in ¹⁶⁴RKKR¹⁶⁷, and cleavageoccurs between ¹⁶⁷RS¹⁶⁸, the amino acid residue numbering is referencedagainst SEQ. ID. NO: 1 minus the 29 amino acid signal peptide. To removethe furin site in order to make a furin-resistant PA, RKKR (residues164-167 of SEQ ID NO: 1 minus the 29 aa signal peptide in SEQ ID NO:1)can be replaced with SSSR (SEQ ID NO: 32) or SSSS (SEQ ID NO: 33) (toeliminate all basic residues). Removal of the furin cleavage site toproduce furin resistant PA (PA^(furin-)) will prevent multimerizationand translocation.

As used herein, “anthrax toxin edema factor” or “EF” refers to acalmodulin- and Ca2+-dependent adenyl cyclase, which elevates the levelof cAMP within the cell. The sequence of EF is known, e.g. NCBI Gene ID:3361726; SEQ ID NO: 6.

As used herein, “anthrax toxin lethal factor” or “LF” refers to ametalloprotease that cleaves most members of the MAP kinase family. Thesequence of LF is known, e.g., NCBI Gene ID: 3361711; SEQ ID NO: 7. Forfurther discussion of EF and LF, see, e.g., Leppla, 79 PNAS, 3162(1982); Duesbery et al., 280 Science 734 (1998); Vitale et al., 248Biochem. Biophys. Res. Commn. 706 (1998); each of which is incorporatedby reference herein in its entirety.

As used herein, “anthrax translocation signal peptide” or “anthrax toxintranslocation peptide” refers to a anthrax-derived domain or peptidethat, when comprised by and/or linked to a polypeptide, causes thatpolypeptide to bind to PA or mPA (optionally to PA or mPA in anoligomeric complex) and be translocated by the mature PA/mPA pore to thecytoplasm of a target cell. In some embodiments, the anthrax toxintranslocation peptide can be LFn (e.g., amino acids 34-267, 34-293, or34-297 of SEQ ID NO:7, the full-length LF with its N-terminal signalpeptide), EFn (e.g., amino acids 59-277 or 34-290 of SEQ. ID. NO:6), orvariants thereof.

In some embodiments of all the aspects described herein, a smallpositively charged peptide segment that mimics LFn or EFn can be used toaid in translocating cargo molecules through a PA or mPA pore. Thesemimics may be composed of at least one non-natural amino acid and aredescribed in more detail in, e.g., International Patent Publication WO2012/096926; U.S. Pat. No. 9,079,952, and US Patent ApplicationPublications No: US 2013/0336974 and US 2015/0267186, each of which isincorporated by reference herein in its entirety.

As used herein, “PAd4” or “protective antigen domain 4” refers to thedomain of PA that recognizes and binds to host cell cellular receptors(e.g. ANTXR1 and/or ANTXR2). PAd4 can comprise the sequence from aboutamino acid 621 to about amino acid 764 of SEQ ID NO: 1 (PAd4 sequenceinclusive of the signal peptide). In some embodiments of all the aspectsdescribed herein, PAd4 can comprise amino acids 621-764 of SEQ ID NO: 1,which is the amino acid sequence of PA with the signal peptide. In someembodiments of all the aspects described herein, PAd4 can comprise aminoacids 596-735 of SEQ ID NO: 1. In one embodiment of all the aspectsdescribed herein, PAd4 can comprise amino acids 625-764 of SEQ ID NO: 1.In another embodiment of all the aspects described herein, PAd4 cancomprise amino acids 616-764 of SEQ ID NO: 1. In another embodiment ofall the aspects described herein, PAd4 can comprise amino acids 609-764of SEQ ID NO: 1. In one embodiment of all the aspects described herein,the PAd4 comprises

(SEQ ID NO: 35) RFHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDEKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG.In one embodiment of all the aspects described herein, PAd4 consistessentially of

(SEQ ID NO: 36) RFHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDEKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG.In one embodiment of all the aspects described herein, the PAd4comprises

(SEQ ID NO: 37) FHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDEKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG.In one embodiment of all the aspects described herein, PAd4 consistessentially of

(SEQ ID NO: 38) FHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDEKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG.In one embodiment of all the aspects described herein, the PAd4comprises

(SEQ ID NO: 39) GLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG.In one embodiment of all the aspects described herein, the PAd4 consistessentially of

(SEQ ID NO: 40) GLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG.

In one embodiment of all aspects of the fusion proteins describedherein, the PAd4 is fused or joined to the other protein or toxin by alinker peptide. Examples of linker peptide include:

(SEQ ID NO: 41) FHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTE, (SEQ ID NO: 42) VEIEDTE, (SEQ ID NO: 43) KDIRKILSGYIVEIEDTE,(SEQ ID NO: 44) STEGLLLNIDKDIRKILSGYIVEIEDTE, and (SEQ ID NO: 45)VGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTE.

Examples of linker peptides attached to the N-terminus of PAd4 are shownas follows where the linker peptide sequences are shown in bold:

(SEQ ID NO: 46) FHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG (SEQ ID NO: 47)VEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG (SEQ ID NO: 48)KDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKK GYEIG (SEQ ID NO: 49)STEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNG IKKILIFSKKGYEIG(SEQ ID NO: 50) VGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG

SNARE-Targeting Toxins (Including BTx and TTx)

Botulinum neurotoxin (BTx, also abbreviated as BoTX or BoNT) causesbotulism which is characterized by descending flaccid paralysis as aresult of inhibition of acetylcholine release at the neuromuscularjunction. There are seven botulinum neurotoxin serotypes (A-G) producedby bacteria of the genus Clostridium. In addition, botulinum-likeneurotoxin from non-Clostridium sp. Weissella oryzae SG25T has recentlybeen discovered (Nature Scientific Repo. Rts|6:30257|DOI:10.1038/srep30257). BTx, with tetanus neurotoxin (TTx) produced byClostridium tetani, make up the clostridial neurotoxin (CNT) family. TTxexhibits a high degree of sequence and structural homology to the BTxs,in particular to BTx/B, and is the causative agent of tetanus, which ischaracterized by spastic paralysis. Although differing in clinicalmanifestation, the fundamental mode of action—inhibition ofneurotransmission—is common to all CNTs. Inhibition of neurotransmitterrelease by the CNTs is caused by the specific cleavage of a group ofproteins integral to the exocytotic process, the SNARE proteins (solubleNSF-attachment protein receptors). Cleavage of one or more of the SNAREproteins leads to a block in the release of vesicular contents to theextracellular environment.

These SNARE targeting toxins share a similar basic CNT structure. CNTsare synthesized as single-chain polypeptides of ˜150 kDa (a holotoxin)and are subsequently cleaved to form di-chain molecules made of thelight (LC) and heavy chains (HC) that are linked by a single disulfidebond. The 50-kDa LC acts as a zinc-dependent endopeptidase. The heavychain contains two functional domains, each of ˜50 kDa. The N-terminalhalf (H_(N)) is the translocation domain, known to form ion channels inlipid bilayers, and the C-terminal half (H_(C)) is the ganglioside andprotein binding domain, which has a key role in binding to the targetcell membrane and internalization of the toxin molecules intocholinergic neurons. The three functional domains are structurallydistinct and arranged in a linear fashion, such that there is no contactbetween the LC and HC domains. Overall, BTxs and TTx share ˜35% sequenceidentity. The BTx catalytic LC domains share up to 36% sequence identity[2], and the LC domains of BTx/B and TTx have over 50% identity. (SeeReview “Botulinum and tetanus neurotoxins: structure, function andtherapeutic utility” by K. Turton et al., Trends in Biochemistry, 2002,27:552-558). Proteases suitable for cleaving the holotoxin to form adi-chain toxin include but are not limited to lysyl peptidase, trypsin,Enterokinase, clostripain, elastase, thermolysin, endoproteinase Lys-C,and endoproteinase Arg-C.

BTx, a neurotoxic protein produced by the bacterium Clostridiumbotulinum, is expressed as a large, single polypeptide molecule that hasthree distinct domains—a 50 kDa proteolytic N-terminal end (LC), a 50kDa translocation domain located in the middle (H_(N)), and a 50 kDahost cell receptor binding C-terminal end (H_(C)). For the toxin to befunctional, it must first be proteolytically cleaved to yield a di-chainprotein consisting of a light chain (LC) and heavy chain(HC=H_(N)+H_(C)), held together by a single disulfide bond. Proteolyticactivation is crucial because after receptor binding and internalisationby endocytosis, subsequent acidification of the endosome is believed tocause a conformational change in the protein, leading to insertion ofthe H_(N) domain into the endosomal membrane, formation of atranslocation pore and delivery of the LC into the cytoplasm, where thedisulphide bond is reduced and the LC released. The LC is azinc-dependant protease with a highly specific substrate specificity.There are multiple BTx serotypes (A to G) and sub-serotypes (up to 12for any given serotype). A serotype is based upon the ability ofneutralising antibodies to neutralise botulinum neurotoxin. To datethere are 7 serotypes of botulinum neurotoxin that have been identified,labelled A through to G (BTx/A to BTx/G).With the advent of nextgeneration sequencing it has been identified that within serotypes thereare subtypes of BoNT, these are defined as toxins with a sequencedifference from other toxins of >2.5% at the protein level. To date over40 subtypes of BoNT have been identified across the seven serotypes.Different serotypes have different substrate specificities—BTx/A andBTx/E cleave SNAP-25, serotypes /B, /D, /F and /G cleavesynaptobrevin/VAMP. BTx/C cleaves both SNAP-25 and syntaxin 1A. Thesesubstrates are SNARE (SNAP (Soluble NSF Attachment Protein) Receptor)proteins that play a critical role in neurotransmitter release at thepre-synaptic nerve terminal and are critical to vesicular secretion fromall eukaryotic cells.

The three domains of BTx (LC, H_(N), H_(C)) are functionally andstructurally distinct and the boundaries of each domain for eachsub-serotype have been defined in the art. (See Review “Botulinum andtetanus neurotoxins: structure, function and therapeutic utility” by K.Turton et al., Trends in Biochemistry, 2002, 27:552-558; this literaturereference is hereby incorporated by reference in its entirety). Each ofthe 50 kDa domains can function independently from each other, forexample, in a chimeric protein. The H_(N) domain has a “belt” regionthat wraps around the LC—this is believed to behave as apseudo-inhibitor and have a chaperone function during LC translocation.The belt regions of the HN of BTx of various serotypes or of TTx areshown in Table 1.

Derivation of botulinum Neurotoxin Sequences

For BTx and TTx molecules with no published structure, the structuralhomology modelling tool LOOPP, available at the website of the loopporganization, was used to obtain a predicted structure based on BTx/A1(3BTA.pdb). From this, as well as a sequence alignment of all BTxsubserotypes by Clustal Omega, it was possible to determine thetransition point between domains. Clustal BTx sequence alignments areprovided at the end of this document for those BTx sero- and sub-typesidentified to date.

LH _(N) =botulinum neurotoxin catalytic domain (LC)+translocation domain(H _(N))

The LH_(N) domain for each subserotype are known in the art, e.g.,LH_(N)/A1 (residues 1-872) and LH_(N)/B1 (residues 1-859).

LC or L=botulinum neurotoxin catalytic domain, (50 kDa, pI ˜6.3-8.1)

The LC domain for each subserotype has previously been defined in US2007/0166332 (hereby incorporated by reference in its entirety), e.g.,LC/A1 (residues 1-448) and LC/B1 (residues 1-441), and are summarized inTable 1 below.

TABLE 1 Neurotoxin Accession Number LC Belt HN BoNT/A1 A5HZZ9 1-448449-546 449-872 BoNT/A2 X73423 1-448 449-546 449-872 BoNT/A3 DQ1859001-444 445-542 445-869 BoNT/A4 EU341307 1-448 449-546 449-872 BoNT/A5EU679004 1-448 449-546 449-872 BoNT/A6 FJ981696 1-448 449-546 449-872BoNT/A7 JQ954969 1-448 449-546 449-872 BoNT/A8 KM233166 1-448 449-546449-872 BoNT/B1 B1INP5 1-440 441-533 441-859 BoNT/B2 AB084152 1-440441-533 441-859 BoNT/B3 EF028400 1-440 441-533 441-859 BoNT/B4 EF0515701-440 441-533 441-859 BoNT/B5 EF033130 1-440 441-533 441-859 BoNT/B6AB302852 1-440 441-533 441-859 BoNT/B7 JQ354985 1-440 441-533 441-859BoNT/B8 JQ964806 1-440 441-533 441-859 BoNT/C1 P18640 1-441 442-542442-867 BoNT/CD AB200360 1-441 442-542 442-867 BoNT/DC AB745660 1-445446-538 446-863 BoNT/D P19321 1-445 446-538 446-863 BoNT/E1 Q00496 1-422423-515 423-846 BoNT/E2 EF028404 1-422 423-515 423-846 BoNT/E3 EF0284031-422 423-515 423-846 BoNT/E4 AB088207 1-422 423-515 423-846 BoNT/E5AB037711 1-422 423-515 423-846 BoNT/E6 AM695759 1-422 423-515 423-846BoNT/E7 JN695729 1-422 423-515 423-846 BoNT/E8 JN695730 1-422 423-515423-846 BoNT/E9 JX424534 1-422 423-515 423-846 BoNT/E10 KF861917 1-422423-515 423-846 BoNT/E11 KF861875 1-422 423-515 423-846 BoNT/E12KM370319 1-425 426-518 426-849 BoNT/F1 Q57236 1-439 440-534 440-865BoNT/F2 GU213209 1-439 440-534 440-865 BoNT/F3 GU213227 1-439 440-534440-865 BoNT/F4 GU213214 1-439 440-534 440-865 BoNT/F5 GU213211 1-438439-531 439-862 BoNT/F6 M92906 1-439 440-534 440-864 BoNT/F7 GU2132331-431 432-524 432-856 BoNT/G Q60393 1-441 442-538 442-864 BoNT/”H”KGO15617 1-434 435-528 435-860 TeNT P04958 1-457 458-556 458-880

The above-identified reference sequences should be considered a guide,as slight variations may occur according to sub-serotypes. By way ofexample, US 2007/0166332 (hereby incorporated by reference in itsentirety) cites slightly different clostridial sequences:

LC (has the Catalytic or Enzymatic Activity Against SNAREs):

Botulinum type A neurotoxin: amino acid residues M1-K448

Botulinum type B neurotoxin: amino acid residues M1-K441

Botulinum type C1 neurotoxin: amino acid residues M1-K449

Botulinum type D neurotoxin: amino acid residues M1-R445

Botulinum type E neurotoxin: amino acid residues M1-R422

Botulinum type F neurotoxin: amino acid residues M1-K439

Botulinum type G neurotoxin: amino acid residues M1-K446

Tetanus neurotoxin: amino acid residues M1-A457

H_(N) Domain:

Botulinum type A neurotoxin: amino acid residues A449-K871

Botulinum type B neurotoxin: amino acid residues A442-S858

Botulinum type C1 neurotoxin: amino acid residues T450-N866

Botulinum type D neurotoxin: amino acid residues D446-N862

Botulinum type E neurotoxin: amino acid residues K423-K845

Botulinum type F neurotoxin: amino acid residues A440-K864

Botulinum type G neurotoxin: amino acid residues S447-S863

Tetanus neurotoxin: amino acid residues S458-V879

Inhibitor Cysteine Knot (ICK) Toxins

As used herein, “inhibitor cysteine knot toxin” or “ICK toxin” refers toa toxin comprising the cysteine knot motif and which modulates theactivity of a receptor and/or ion channel target. An inhibitor cysteineknot (ICK) is a protein structural motif containing three disulfidebridges. Along with the sections of polypeptide between them, twodisulfides form a loop through which the third disulfide bond (linkingthe 3rd and 6th cysteine in the sequence) passes, forming a knot (thusthe alternate name knottin). The motif is common in invertebrate toxinssuch as those from arachnids and molluscs. The motif is also found insome inhibitor proteins found in plants, but the plant and animal motifsare thought to be a product of convergent evolution. The ICK motif is avery stable protein structure which is resistant to heat denaturationand proteolysis. ICK peptide components of venoms target voltage-gatedion channels but members of the family also act as antibacterial andhaemolytic agents. Plant ICK proteins are often protease inhibitors. ICKtoxins are typically found in the venom of, e.g., cone snails, spiders,and scorpions. In some embodiments, ICK toxins are disulfide-containingpeptide toxins. These disulfide-containing peptide toxins have between30-70 amino acid residue. In some embodiments of any of the aspectsdescribed herein, the ICK toxin is a conotoxin, an agatoxin, adelta-palutoxin, a huwentotoxin or a ProTx II toxin.

Huwentotoxins are 7 types of ICK toxins (HWTX-1, HWTX-III, HWTX-IV,HWTX-X, HWTX-II, HWTX-VII, HWTX-VIII) from Chinese bird spiders that actagainst voltage-gated calcium channels.

Delta-palutoxins consist of 4 types of ICK Toxins (IT1, IT2, IT3, IT4)from spiders that act against voltage-gated sodium channels.

Conotoxins are small, 10-30 residue peptide ICK toxins from cone snailswhich act on voltage gated calcium and sodium channels. Some of thesetoxins are known to act extracellularly to modulate the activity of ionchannels. Examples, W-conotoxin GVIA and W-conotoxin MVIIC.

As used herein, “conotoxin” refers to a toxin produced by the marinecone snail (e.g. the genus Conus). Some conotoxins can modulate ionchannel activity. In some embodiments of all the aspects describedherein, the conotoxin can be an ion channel modulator. Non-limitingexamples of conotoxins can include δ-conotoxin (e.g. NCBI ID: AKD43185;SEQ ID NO: 10; for further discussion see, e.g., Leipold et al. FEBSLetters 2005 579:3881-4, which is incorporated by reference herein inits entirety), which is known to block voltage-dependent sodiumchannels, μ-conotoxin (e.g. Swiss-Prot ID: P15472.1; SEQ ID NO: 9; forfurther discussion, see, e.g., Li and Tomaselli. Toxicol. 200444:117-122; which is incorporated by reference herein in its entirety)which also blocks voltage-dependent sodium channels, or ω-conotoxin MVII A (e.g. NCBI ID: ADB93081; SEQ ID NO: 8; for further discussion see,e.g., Nielsen et al. Molecular Recognition 2000 13:55-70, which isincorporated by reference herein in its entirety) (e.g. ziconotide),which is known to block N-type voltage-dependent calcium channels.Additional non-limiting examples of ICK toxins include, e.g.,psalmotoxin-1, β-TRTX-Tp2a, and purotoxin-1 and are described in theliterature (see, e.g., US Patent Publications 20120277166; 20120220539;20120087969, 20050214903, and 20050143560; Craik et al. Toxicon 200139:43-60; Zhu et al. FASEB Journal 2003 17:1765-7; Daly and Craik.Current Opinion in Chemical Biology 2011 15:362-368; Grishin. EuropeanJournal of Biochemistry 1999 264: 276-280; Liang et al. Toxicon 200443:575-585; Kolmar FEBS Journal 2008 275: 2684-2690; Saez et al. Toxins2010 2:2851-2871; Vetter et al. Amino Acids 2011 40:15-28; Alewood etal. Australian Journal of Chemistry 2003 56:769-774; King. ExpertOpinion on Biological Therapy 2011 11:1469-1484; King et al. Toxicon2008 52:264-276; Herzig et al. Nucl. Acids Res 2010; Szeto et al. FEBSLetters 2000 470: 203-299; and Bergeron and Bingham. Toxins 20124:1082-1119; each of which is incorporated by reference herein in itsentirety).

The following are examples of cysteine knot sequences from ICK toxinsthat can attach to PAd4, mPA, PA^(furin-), LFn, EFn, or othernociceptor-binding protein etc for delivery to nociceptor neurons.

W-conotoxin GVIA: (SEQ ID NO: 51) CKSXGSSCSXTSYNCCRSCNXYTKRCY(Modifications: X = Hyp, Disulfide bridgebetween 1-16, 8-19, 15-26, Tyr-27 = C-terminal amide) W-conotoxin MVIIC:(SEQ ID NO: 52) CKGKGAPCRKTMYDCCSGSCGRRGKC(Modifications: Disulfide bridge between 1-16,8-20, 15-26, Cys-26 = C-terminal amide) W-Agatoxin IVA: (SEQ ID NO: 53)KKKCIAKDYGRCKWGGTPCCRGRGCICSIMGTNCECKPRLIMEGLGLA(Modifications: Disulfide bridge between 4-20, 12-25, 19-36, 27-34)W-Agatoxin TK: (SEQ ID NO: 54)EDNCIAEDYGKCTWGGTKCCRGRPCRCSMIGTNCECTPRLIMEGLSFA(Modifications: Disulfide bridge between 4-20,  12-25, 19-36, 27-34)Huwentotoxin IV: (SEQ ID NO: 55) ECLEIFKACNPSNDQCCKSSKLVCSRKTRWCKYQI(Modifications: Disulfide bridge: 2-17, 9-24,16-31)(Modifications: Ile-35 = C-terminal amide) ProTx II: (SEQ ID NO: 56) YCQKWMWTCDSERKCCEGMVCRLWCKKKLW (Modifications: Disulfide bridge: 2-16, 9-21,  15-25)

The AB Toxins

The AB toxins are two-component protein complexes secreted by a numberof pathogenic bacteria. They are named AB toxins due to theircomponents: the “A” component is usually the “active” portion, and the“B” component is usually the “binding” portion. The “A” subunitpossesses enzyme activity, where the catalytic domain or activity isfound, and is transferred to the host cell following a conformationalchange in the membrane-bound transport “B” subunit. Among the toxinsproduced by certain Clostridium spp. are the binary exotoxins. Theseproteins consist of two independent polypeptides, which correspond tothe A/B subunit moieties. The enzyme component (A) enters the cellthrough endosomes produced by the oligomeric binding/translocationprotein (B), and prevents actin polymerisation through ADP-ribosylationof monomeric G-actin.

Examples of the “A” component of the binary toxin family include C.perfringens iota toxin Ia, C. botulinum C2 toxin CI, and Clostridiumdifficile ADP-ribosyltransferase. Other homologous proteins have beenfound in Clostridium spiroforme.

Examples of the of the “B” component (aka binding or transportcomponent) binary toxin family include the Bacillus anthracis protectiveantigen (PA) protein described herein.

The Diphtheria toxin (DT) also is an AB toxin. It inhibits proteinsynthesis in the host cell through phosphorylation of the eukaryoticelongation factor 2, which is an essential component for proteinsynthesis. The exotoxin A of Pseudomonas aeruginosa is another exampleof an AB toxin that targets the eukaryotic elongation factor 2.

Engineered Fusion Chimeric Proteins

BTx/TTx—PAd4/PA Fusion Proteins

Encompassed herein is a BTx or TTx fused to PAd4 to allow redirectingthe action of TTx or one or another BTx to nociceptors via PAd4 binding.This encompasses a BTx in which the receptor binding domain is replacedby the receptor binding function of anthrax PA. In this construct wereplace the C-terminal domain of BTx with the C-terminalreceptor-binding domain of PA, PAd4 or PA63, or any PA fragment that canstill bind the receptor ANTXR2. The BTx Light chain (enzymatic moiety isencompassed in the L-chain) and translocation domain (H_(N)) are linkedto a receptor binding domain of PA. (See FIG. 7A) The C-terminalreceptor-binding domain of PA, e.g., PAd4, will bind theconstruct/fusion protein to the ANTXR2 receptor on nociceptors andmediate trafficking to the endosome, at which point the BTx pore formingdomain (H_(N)) would insert into the membrane and mediate translocationof the BTx L-chain to the cytosol, where it would cleave a SNARE andblock neurotransmitter release. PA will target its receptor ANTXR2(CMG2), and botulinum toxin H_(N) domain will trigger pore-formation,translocation, and enzymatic moieties to block synaptic function. Insome embodiments, this strategy requires botulinum or tetanus toxins toact both extracellularly and intracellularly. In some embodiments, thisstrategy requires the pre-activation of the toxins by Lys-C enzyme. Thisapproach could apply similarly to the translocation domain in the heavychain and light chain domains of tetanus toxin. (See FIG. 7B). Otherproteases suitable for cleaving the junction include but are not limitedto lysyl peptidase, trypsin, Enterokinase, clostripain, elastase,thermolysin, endoproteinase Lys-C, and endoproteinase Arg-C.

Accordingly, in one aspect, the engineered fusion protein comprises (a)PAd4 and (b) a BTx or TTx, wherein the PAd4 is fused or linked with theBTx or TTx. BTx or TTx are SNARE-targeting proteases. In one embodiment,the engineered fusion protein comprises (a) a PAd4 and (b) aSNARE-targeting protease, wherein the PAd4 is fused or linked with theSNARE-targeting protease. In one embodiment of the engineered fusionprotein, the PAd4 domain is replaced with PA. In one embodiment, the PAis a variant PA mutant form that is resistant to cleavage by furin. Inanother aspect, we provide a composition comprising an engineered fusionprotein comprises (a) a PAd4 and (b) a SNARE-targeting protease, whereinthe PAd4 is fused or linked with the SNARE-targeting protease. In oneembodiment, provided herein is a composition comprising an engineeredfusion protein that comprises (a) PAd4 and (b) a BTx or TTx, wherein thePAd4 is fused or linked with the BTx or TTx. In another embodiment ofthe composition, the PAd4 domain of the engineered fusion proteinincludes PA, instead of PAd4. In one embodiment, the PA is a variant PAmutant form that is resistant to cleavage by furin. In anotherembodiment of any of the fusion polypeptide compositions describedherein, the composition further comprises a pharmaceutically acceptablecarrier or excipient.

In one embodiment of a fusion protein or composition described herein,the PAd4 or PA is linked with the TTx, BTx, or SNARE targeting proteasewith a peptide linker. In one embodiment of a fusion protein orcomposition, the linker peptide is 1-20 amino acids long. In oneembodiment of a fusion protein or composition, the PAd4 or PA canreplace the native receptor binding domain of the BTx or TTx neurotoxin,or is fused to a form of one of the BTx or TTx neurotoxins in which thenative receptor-binding function had been ablated by mutation. In oneembodiment of a fusion protein or composition, the TTx or BTx orSNARE-targeting protease comprises the entire protein, i.e., theholotoxin, wherein the native receptor-binding function had been ablatedby mutation. In one embodiment of a fusion protein or composition, theTTx or BTx or SNARE-targeting protease consist essentially of the entireprotein, i.e., the holotoxin, wherein the native receptor-bindingfunction had been ablated by mutation. In one embodiment of a fusionprotein or composition, the TTx or BTx or SNARE-targeting proteaseconsists of the entire protein, i.e., the holotoxin, wherein the nativereceptor-binding function had been ablated by mutation. In oneembodiment of a fusion protein or composition, the TTx or BTx orSNARE-targeting protease comprises, consist essentially of, or consistsof only a part of the protein, rather than the holotoxin, e.g., one ortwo domains of the holotoxin protein. For example, the TTx or BTx orSNARE-targeting protease element can consist essentially of the LC andH_(N) (LH_(N)) of a TTx, BTx or SNARE-targeting protease.

In another aspect, described herein is a fusion protein comprising (a) abotulinum neurotoxin (BTx) moiety comprising an N-terminal enzymaticdomain (LC), and (b) an intermediate pore-forming/translocation-domain(H_(N)) of the BTx, and (c) a C-terminal receptor-binding domain ofanthrax toxin protective antigen (PAd4 domain), wherein the parts(a)-(c) are linked together, e.g., linked together by linker peptides asdescribed herein. In other words, an L H_(N) of a BTx is fused to a PAd4domain of PA by a linker peptide, wherein PAd4 is the C-terminalreceptor-binding domain of anthrax toxin protective antigen. In oneembodiment of a fusion protein, PA is included instead of the PAd4. Inone embodiment, the PA is a variant PA mutant form that is resistant tocleavage by furin. In another aspect, described herein is a compositioncomprising a fusion protein comprising (a) a botulinum neurotoxin (BTx)moiety comprising an N-terminal enzymatic domain (LC), and (b) anintermediate pore-forming/translocation-domain (H_(N)) of the BTx, and(c) a C-terminal receptor-binding domain of anthrax toxin protectiveantigen (PAd4 domain), wherein the parts (a)-(c) are linked together. Inone embodiment, the composition includes PA in place of the PAd4 domainin the fusion protein. In another embodiment, the PA is a variant PAmutant form that is resistant to cleavage by furin. In anotherembodiment of all the aspects described herein, a composition asdescribed herein can further comprise a pharmaceutically acceptablecarrier or excipient.

In one embodiment of a fusion protein or composition described herein,the BTx moiety is selected from the BTx light chain (LC) and heavy chain(HC) domains of any one of Clostridial BTx serotypes: BTx/A, BTx/B,BTx/C, BTx/D, BTx/E, BTx/F, BTx/G, and a non-Clostridial botulinum-liketoxin.

In another aspect, described herein is a fusion protein comprising (a)an N-terminal enzymatic domain (LC) of tetanus neurotoxin (TTx), (b) atranslocation/pore-forming domain (H_(N)) of TTx, and (c) a C-terminalreceptor-binding domain of anthrax toxin protective antigen (PAd4domain), wherein the parts (a)-(c) of fusion protein are linkedtogether, e.g., operably linked together by linker peptides describedherein. In other words, a LH_(N) of aTTx is fused to to a PAd4 domain ofPA by a linker peptide, wherein PAd4 is the C-terminal receptor-bindingdomain of anthrax toxin protective antigen. In one embodiment, describedherein is a composition comprising a fusion protein comprising (a) anN-terminal enzymatic domain (LC) of tetanus neurotoxin (TTx), (b) atranslocation/pore-forming domain (H_(N)) of TTx, and (c) a C-terminalreceptor-binding domain of anthrax toxin protective antigen (PAd4domain), wherein the parts (a)-(c) of the fusion protein are linkedtogether. In some embodiments of all the aspects described herein, acomposition as described herein can further comprise a pharmaceuticallyacceptable carrier or excipient. In another embodiment of a fusionprotein or composition, the amino acid residues corresponding to thejunction between the light chain of TTx and the heavy chain of TTx hasbeen cleaved.

In one embodiment of a fusion protein or composition described herein,the amino acid residues corresponding to the LC junction with the HC ofthe BTx (serotypes included) or with TTx has been cleaved. In oneembodiment of a fusion protein or composition described herein, theamino acid residues corresponding to the LC junction with the H_(N) ofthe BTx (serotypes included) or TTx has been cleaved. The cleavage iscarried out by a protease, such as but is not limited to, lysylpeptidase, trypsin, Enterokinase, clostripain, elastase, thermolysin,endoproteinase Lys-C, and endoproteinase Arg-C.

In one embodiment of any of the fusion proteins including a BTx moiety,the BTx moiety comprises a BTx or TTx enzymatic moiety and atranslocation peptide/domain. In one embodiment, the enzymatic moietyand a translocation peptide/domain are linked by a linker peptide. Inone embodiment of any of the fusion proteins including a BTx moietycomprising a BTx or TTx enzymatic moiety and a translocationpeptide/domain, the enzymatic moiety and a translocation peptide/domainare separated by cleavage with a protease, e.g., Lys-C. In oneembodiment of any of the fusion proteins including a BTx moietycomprising a BTx or TTx enzymatic moiety and a translocationpeptide/domain linked by a linker peptide, the enzymatic moiety and atranslocation peptide/domain are separated by cleavage, e.g., with aprotease, e.g., Lys-C. Cleavage functions to activate the enzymaticmoiety and a translocation peptide/domain in the fusion protein.

In one embodiment of any of the fusion proteins including a BTx moiety,wherein the BTx moiety comprises, consist essentially of, or consists ofan L chain and a H_(N) domain of the BTx, the S-S bridge between the Lchain and the H_(N) domain is not reduced. In one embodiment of any ofthe fusion proteins including a BTx moiety, wherein the BTx moietycomprises, consist essentially of, or consists of an L chain and not aH_(N) domain of the BTx, the Cys residues in the L-chain and the beltcorresponding to the N-terminal part of the BTx H_(N) domain in theholotoxin, if present, can be changed to Ala, Ser, or Thr. In oneembodiment of a fusion protein or composition described herein, thefusion protein further comprises at least one D-amino acid at theN-terminus of the fusion protein. The D-amino acid residues in theN-terminus serve to ]decrease potential immunogenicity of the fusionprotein.

In one embodiment of a fusion protein or composition described herein,the PAd4 is linked with the LH_(N) with a peptide linker. In oneembodiment, the linker peptide is 1-20 amino acids long. In oneembodiment, about 1-60 consecutive amino acids from the N-terminal sideof PA adjacent to the native PAd4 domain are further incorporatedbetween the enzymatic/pore-forming domains of the fusion protein and thereceptor binding PAd4 fusion partner. In one embodiment of fusionprotein or composition, the PAd4 is located at the C-terminus of thefusion protein, and the LH_(N) is at the N-terminus of the fusionprotein. In one embodiment of a fusion protein or composition, the PAd4is located at the N-terminus of the fusion protein. In anotherembodiment of a fusion protein or composition, the PAd4 is located bothat the N-terminus and the C-terminus of the fusion protein, with theLH_(N) (LC and H_(N)) sandwiched between two PAd4 domains. In anotherembodiment of a fusion protein or composition, there are more than onePAd4 domain in the fusion protein, e.g., two to ten PAd4 domains, one tofive PAd4 domains, or two to five PAd4 domains. In one embodiment of afusion protein or composition described herein, where multiple PAd4domains are present, the multiple PAd4 domains are arranged in tandem.In one embodiment of a fusion protein or composition described herein,the multiple PAd4 domains are linked by peptide linkers. In oneembodiment, the linker peptide is 1-20 amino acids long.

BTx/TTx—LFn/EFn Fusion Proteins

Native PA and a fusion protein comprising LFn fused to the catalyticdomain of TTx or the catalytic domain of one or another of the variousforms/serotypes of BTx, when used in combination can be directed todisrupt intracellular signaling in the nociceptor neurons or blocksynaptic transmission via neurotransmitters. (See FIGS. 8A and 8B) Theseconstructs make use of the proteolytic activities of the catalyticdomains to cleave SNARE proteins and thereby block neurotransmitterrelease without killing nociceptors or bystander cells. The TTx or oneor another of the various forms/serotypes of BTx or CNT family of toxinsare SNARE-targeting proteases. Accordingly, in one aspect, theengineered fusion protein comprises (a) an LFn and (b) a SNARE-targetingprotease. In one embodiment, the engineered fusion protein comprises (a)an LFn and (b) a TTx or a BTx. In one embodiment of a fusion proteindescribed herein, LFn is linked with the TTx, BTx, or SNARE targetingprotease with a peptide linker. In one embodiment, provided herein is acomposition comprising a engineered fusion protein comprising (a) an LFnand (b) a TTx or a BTx. It one embodiment, provided herein is acomposition comprising an engineered fusion protein comprising (a) anLFn and (b) a SNARE-targeting protease. In one embodiment of all theaspects described herein, a fusion protein composition can furthercomprise a pharmaceutically acceptable carrier or excipient. In oneembodiment of a fusion protein or composition described herein, LFn islinked with the TTx, BTx, or SNARE targeting protease with a peptidelinker. In one embodiment, the linker peptide is 1-20 amino acids long.In one embodiment of an engineered fusion protein or compositiondescribed herein, the LFn is located at the N-terminus of the fusionprotein. In one embodiment of an engineered fusion protein orcomposition described herein, the LFn is located at the C-terminus ofthe fusion protein. In one embodiment of an engineered fusion proteinsdescribed herein, the LFn is located both at the N-terminus andC-terminus of the fusion protein, with the TTx or BTx or SNARE-targetingprotease sandwiched between the two LFns. In another embodiment of anengineered fusion protein or composition described herein, EFn isincluded instead of LFn. In one embodiment of a fusion protein orcomposition described herein, the TTx or BTx or SNARE-targeting proteasecomprises the entire protein, i.e., the holotoxin. In one embodiment ofa fusion protein or composition, the TTx or BTx or SNARE-targetingprotease consist essentially of the entire protein, i.e., the holotoxin.In one embodiment of a fusion protein or composition, the TTx or BTx orSNARE-targeting protease consists of the entire protein, i.e., theholotoxin. The fusion protein containing the holotoxin would need to beactivated by proteolytic cleavage. Proteases suitable activating theholotoxin include but are not limited to lysyl peptidase, trypsin,Enterokinase, clostripain, elastase, thermolysin, endoproteinase Lys-C,and endoproteinase Arg-C. Proteases suitable for cleaving the junctioninclude but are not limited to lysyl peptidase, trypsin, Enterokinase,clostripain, elastase, thermolysin, endoproteinase Lys-C, andendoproteinase Arg-C. In one embodiment of a fusion protein orcomposition, the TTx or BTx or SNARE-targeting protease comprises,consist essentially of, or consists of only a part of the protein, notthe holotoxin, e.g., a domain of the holotoxin. For example, the TTx orBTx or SNARE-targeting protease can consist essentially of LC or LC andH_(N) (LH_(N)) of the TTx or BTx or SNARE-targeting protease. Forexample, the TTx or BTx or SNARE-targeting protease can consistessentially of LC plus the belt segment located at the N-terminus of theholotoxin, the belt segment is found between the L chain and the H_(N)chain. In one embodiment of a fusion protein or composition described,the BTx is selected from the BTx light chain (LC) and heavy chain (HC)domains of any one of serotypes: BTx/A, BTx/B, BTx/C, BTx/D, BTx/E,BTx/F, BTx/G, and a non-Clostridial botulinum-like toxin. In oneembodiment of a fusion protein or composition described, the BTx isselected from BTx light chain (LC) and heavy chain (HC) domains fromTable 1, as non-limiting examples, see SEQ. ID. NOS: 29-31.

Accordingly, in one aspect, provided herein is a fusion proteincomprising (a) an enzymatic moiety of a botulinum neurotoxin (BTx) or anan enzymatic moiety of a tetanus neurotoxin (TTx), and (b)(i) theN-terminal domain (LFn) of anthrax toxin lethal factor; or (b)(ii) theN-terminal domain (EFn) of anthrax toxin edema factor. The enzymaticmoiety of BTx is located at the N-terminal of botulinum neurotoxinholotoxin. In one embodiment, provided herein is a compositioncomprising a fusion protein comprising (a) an enzymatic moiety of abotulinum neurotoxin (BTx) or an an enzymatic moiety of a tetanusneurotoxin (TTx), and (b)(i) the N-terminal domain (LFn) of anthraxtoxin lethal factor; or (b)(ii) the N-terminal domain (EFn) of anthraxtoxin edema factor. In one embodiment of all the aspects describedherein, the composition further comprises a pharmaceutically acceptablecarrier or excipient.

In one aspect, provided herein is a fusion protein comprising: (a) anon-cytotoxic protease, which protease is capable of cleaving a SNAREprotein in a nociceptor neuron; and (b) a protein capable of binding toan anthrax toxin protective antigen (PA) or a fragment thereof, whereinthe PA or PA fragment binds a receptor expressed on the nociceptorneuron. In one embodiment, provided herein is a fusion proteincomprising: (a) a non-cytotoxic protease, which protease is capable ofcleaving a SNARE protein in a nociceptor neuron; and (b) a proteincapable of binding to an anthrax toxin protective antigen (PA) or a PAfragment thereof, wherein the PA or PA fragment binds a receptorexpressed on the nociceptor neuron. In some embodiments of all theaspects described herein, the composition as described herein furthercomprise a pharmaceutically acceptable carrier or excipient.

In one embodiment of a fusion protein or composition as describedherein, the non-cytotoxic protease comprises a clostridial neurotoxinL-chain (LC) or a non-Clostridial botulinum-like toxin L-chain. In oneembodiment of a fusion protein or composition, the clostridialneurotoxin L-chain (LC) or a non-Clostridial botulinum-like toxinL-chain is the enzymatic moiety. In one embodiment of a fusion proteinor composition, the non-cytotoxic protease is selected from the groupconsisting of the BTx light chain domains of any one of BTx/A, BTx/B,BTx/C, BTx/D, BTx/E, BTx/F, BTx/G, a first non-Clostridialbotulinum-like toxin, and a TTx. For example, the non-cytotoxic proteasecan be selected from the group consisting of BTx/A LC (a.a. 1-448),BTx/B LC (a.a. 1-441), BTx/C LC (a.a. 1-449), BTx/D LC (a.a. 1-442),BTx/E LC (a.a. 1-422), BTx/F LC (a.a. 1-436), and BTx/GLC (a.a. 1-442).For examples, the the BTx light chain can be selected from SEQ ID NO:20-28 or Table 1 described herein.

In one embodiment of a fusion protein or composition, the clostridialneurotoxin is BTx or TTx.

In one embodiment of a fusion protein or composition, the PA-bindingreceptor expressed on the nociceptor neuron is ANTXR2 (CMG2).

In one embodiment of a fusion protein or composition, the proteincapable of binding to PA is: (i) an anthrax toxin lethal factor (LF); or(ii) an anthrax toxin edema factor (EF).

In one embodiment of a fusion protein or composition, the PA bindingdomain of LF is the N-terminal domain of LF, (abbreviated as LFPABD orLFn).

In one embodiment of a fusion protein or composition, the PA bindingdomain of EF is the N-terminal domain of EF, (abbreviated as EFPABD orEFn).

In one embodiment of a fusion protein or composition, the LFn and EFnare domains that bind to oligomeric forms of PA63, the proteolyticallyactivated form of anthrax PA. In one embodiment of a fusion protein orcomposition, the enzymatic domain is the LC of the BTx or TTx describedherein. In one embodiment of a fusion protein or composition, theenzymatic domain is linked N-terminally or C-terminally or bothN-terminally and C-terminally, to LFn or EFn. In one embodiment of afusion protein or composition described herein, the LFn or EFn is linkedwith the enzymatic domain or the LC of the BTx or TTx by way of apeptide linker. In one embodiment, the linker peptide is 1-20 aminoacids long. In one embodiment of an engineered fusion protein orcomposition described herein, the LFn or EFn is located at theN-terminus of the fusion protein. In one embodiment of an engineeredfusion protein or composition described herein, the LFn or EFn islocated at the C-terminus of the fusion protein. In one embodiment of anengineered fusion protein or composition described herein, the LFn orEFn is located both at the N-terminus and C-terminus of the fusionprotein, with the N-terminal enzymatic domain or the LC of the BTx orTTx sandwiched between the two LFns or EFns. In one embodiment of afusion protein or composition described herein, the fusion protein canfurther comprise an amino acid sequence defining a belt corresponding tothe N-terminal part of the BTx H_(N) domain, wherein the H_(N) domain islocated at the C-terminal side of the BTx.

In one embodiment of a fusion protein or composition described herein,where the fusion protein comprises both the L and H_(N) of a BTx or TTx,e.g., amino acids 1-872 (SEQ. ID. NO:29), the amino acid residuescorresponding to the LC junction with the H_(N) of BTx have beencleaved. Proteases suitable for cleaving the junction include but arenot limited to lysyl peptidase, trypsin, Enterokinase, clostripain,elastase, thermolysin, endoproteinase Lys-C, and endoproteinase Arg-C.In one embodiment of any of the fusion proteins including a BTx moiety,wherein the BTx moiety comprises, consist essentially of, or consists ofan L chain and a H_(N) domain of BTx, the S-S bridge between the L chainand the H_(N) domain is not reduced. In one embodiment of any of thefusion proteins including a BTx moiety, wherein the BTx moietycomprises, consist essentially of, or consists of an L chain and not aH_(N) domain of BTx, the Cys residues in the L-chain and the beltcorresponding to the N-terminal part of the BTx H_(N) domain in theholotoxin, if present, have been changed to Ala, Ser, or Thr. In oneembodiment of a fusion protein or composition described herein, thefusion protein further comprises at least one D-amino acid at theN-terminus of the fusion protein. The D-amino acid can be added by aSortase reaction and are described in more detail in, e.g.,International Patent Publication WO 2012/096926; U.S. Pat. No.9,079,952, and US Patent Application Publications No: US 2013/0336974and US 2015/0267186, each of which is incorporated by reference hereinin its entirety.

In one embodiment of a fusion protein described herein that comprises anLFn or EFn, or LF or EF, these fusion proteins are used together withnon-fused PA, or used together with a second fusion protein comprising(a) PA or PA fragment capable of binding LFn or EFn, and (b) anociceptor neuron-binding protein, where the PA or PA fragment is fusedto a nociceptor neuron-binding protein, and the nociceptorneuron-binding protein directs the toxin to nociceptor neurons to treatpain. In other words, in one embodiment, the fusion protein describedherein comprising an LFn or EFn, or LF or EF is co-administered with aseparate, non-fused PA polypeptide to a subject to treat pain. Inanother embodiment, the fusion protein described herein comprising anLFn or EFn, or LF or EF is co-administered with a second fusion proteinto a subject to treat pain. The second fusion protein comprises (a) PAor a PA fragment capable of binding LFn or EFn, and (b) a nociceptorneuron-binding protein, where the PA or PA fragment is fused to anociceptor neuron-binding protein, and the nociceptor neuron-bindingprotein directs the first fusion protein containing the toxin tonociceptor neurons to treat pain. In some embodiments of the secondfusion protein, the PA is a variant (mPA), a modified or mutated formthat does not bind the ANTXR2 receptor as described herein.

In one aspect, then, provided herein is a composition comprising:

(I) a first fusion protein comprising (a) a botulinum neurotoxinN-terminal enzymatic domain of a botulinum neurotoxin (BTx) moiety, theenzymatic domain, or tetanus neurotoxin (TTx) moiety, the enzymaticdomain and (b)(i) the N-terminal domain (LFn) of anthrax toxin lethalfactor or (b)(ii) the N-terminal domain (EFn) of anthrax toxin edemafactor, and

(II) a second protein comprising (c) PA or PA fragment capable ofbinding LFn or EFn, and optionally (d) nociceptor neuron-bindingprotein,

wherein the parts (a) and (b) are joined by a peptide linker, andwherein the parts (c) and (d) are also joined by a peptide linker, whenpart (d) is present. In one embodiment of the composition described,when the second protein is a fusion protein, the PA is a mutant variantof PA, an mPA.

In one embodiment of any of the toxin proteins, toxin fusion proteins orfusion protein compositions described herein, the composition furthercomprises a pharmaceutically acceptable carrier or excipient. In oneembodiment of a composition comprising a linker peptide, the linkerpeptide is 1-20 amino acids long. An non-limiting example of a PAfragment capable of binding LFn or EFn is PA63. In one embodiment, thePA protein is an oligomeric PA. In one embodiment, the PA is a nativeanthrax toxin protective antigen (PA) protein. In one embodiment of acomposition described herein, the PA is an oligomeric PA, which can bebound to the fusion protein. In one embodiment, this composition isuseful for the treatment of pain such as nerve, joint, skin, visceral,bladder, or muscle pain, and diabetic neuropathic pain, cancer pain,fibromyalgia or other systemic pain disorders. In another embodiment,this composition is useful in the manufacture of medicament for thetreatment of pain.

AB Toxin—LFn/EFn Fusion Proteins

Native PA and a fusion protein LFn-DT in combination can be directed tonociceptor neurons using the catalytic domain (aka the “A” component or“A” part) of diphtheria toxin (DT) to block protein synthesis. DT is anAB type toxin. Accordingly, in one aspect, an engineered fusion proteincomprises (a) an LFn and (b) a DT. In one embodiment, provided herein isa composition comprising an engineered fusion protein comprising (a) anLFn and (b) a DT. In one embodiment of a fusion protein, LFn is linkedto the DT with a peptide linker. In one embodiment of a fusion protein,DT comprises both the A part and the B part found in SEQ. ID. NO: 2. Inone embodiment, DT is DTA, comprising the A part (the active,catalytic/enzymatic domain) found in SEQ ID NO: 2. The DTA amino acidsequence includes residues 1-193 of the diphtheria toxin. In oneembodiment of an engineered fusion protein comprising LFn, the LFn islocated at the N-terminus of the fusion protein. In another embodimentof an engineered fusion protein comprising LFn, the LFn is located atthe C-terminus of the fusion protein. In another embodiment of anengineered fusion protein comprising LFn, the LFn is located both at theN-terminus and C-terminus of the fusion protein, with DT sandwichedbetween the two LFns. In another embodiment, the LFn is replaced withEFn. Accordingly, in one embodiment, the engineered fusion proteincomprises (a) an EFn and (b) a DT. In one embodiment, provided herein isa composition comprising an engineered fusion protein comprising (a) anEFn and (b) a DT. In some embodiments of all the aspects describedherein, a composition as described herein can further comprise apharmaceutically acceptable carrier or excipient.

In some embodiments of an engineered fusion protein or compositiondescribed herein, the catalytic domains of other intracellularly actingtoxins, such as the plant toxin, ricin, or a disulfide-containingpeptide toxin such as the ICK toxin could be fused to LFn in place ofDT, giving an engineered fusion protein such as LFn-PE (PTx),LFn-conotoxin, LFn-ricin, LFn-Cholera toxin, LFn-agatoxin,LFn-delta-palutoxin, LFn-huwentotoxin, LFn-scorpion long-chain toxin,and/or LFn-Shiga toxin. Accordingly, in one embodiment of an engineeredfusion protein or composition described herein, the LFn is linked withone other toxin, the other toxin being an intracellularly acting toxin.In one embodiment of a fusion protein or composition, theintracellularly acting toxin is selected from the group consisting ofricin, PE, conotoxin, Cholera toxin, agatoxin, delta-palutoxin,huwentotoxin, Shiga toxin, scorpion long-chain toxin, and scorpionshort-chain toxin. In one embodiment of a fusion protein or composition,LFn is linked to the other toxin with a peptide linker. In anotherembodiment of an engineered fusion protein or composition describedherein, the LFn is linked with at least one other toxin that is notderived from the anthrax toxin. In another embodiment of an engineeredfusion protein or composition described herein, the LFn is linked to theA component or active, catalytic or enzymatic domain of an AB toxin,e.g., the A component residues between amino acid residues 364-613 ofPE, the A part residues are 1-193 of the DT.

In one embodiment of an engineered fusion protein or compositiondescribed herein, LFn is located at the N-terminus of the fusionprotein. In another embodiment of an engineered fusion protein orcomposition described herein, LFn is located at the C-terminus of thefusion protein. In one embodiment of an engineered fusion protein orcomposition described herein, LFn is located both at the N-terminus andC-terminus of the fusion protein, with the intracellularly acting toxinsandwiched between the two LFns. In another embodiment of an engineeredfusion protein or composition described herein, EFn is included insteadof LFn. For example, this provides an engineered fusion protein such asEFn-PE (PTx), EFn-conotoxin, EFn-ricin, EFn-Cholera toxin, EFn-agatoxin,EFn-delta-palutoxin EFn-huwentotoxin, or EFn-Shiga toxin.

In some embodiments of a fusion protein described herein comprising aLFn or EFn, or LF or EF, these fusion proteins are used together withnon-fused or separate PA, or used together with a second fusion proteinto treat pain. In other words, in one embodiment, the fusion proteindescribed herein comprising an LFn or EFn, or LF or EF polypeptide isco-administered with PA to a subject to treat pain. In anotherembodiment, the fusion protein described herein comprising an LFn orEFn, or LF or EF is co-administered with a second fusion protein to asubject to treat pain. The second fusion protein comprises (a) PA or PAfragment capable of binding LFn or EFn, and (b) a nociceptorneuron-binding protein, where the PA or PA fragment is fused to anociceptor neuron-binding protein, and the nociceptor neuron-bindingprotein directs the first fusion protein containing the toxin tonociceptor neurons to treat pain. In some embodiments of the secondfusion protein, the PA is a mutant variant of the native PA (mPA), aform that does not bind the ANTXR2 receptor as described herein.

In one aspect, provided herein is a composition comprising:

(I) a first fusion protein comprising (a) a DT, an intracellularlyacting toxin, an ICK toxin or a disulfide-containing peptide toxin, and(b)(i) the N-terminal domain (LFn) of anthrax toxin lethal factor or(b)(ii) the N-terminal domain (EFn) of anthrax toxin edema factor, and

(II) a second protein comprising (c) PA or PA fragment capable ofbinding LFn or EFn, and optionally (d) a nociceptor neuron-bindingprotein,

wherein the parts (a) and (b) are joined by a peptide linker, and

wherein the parts (c) and (d) are also joined by a peptide linker when(d) is present. In one embodiment of the composition described, when thesecond protein is a fusion protein, the PA is a mutant variant of PA, amPA.

In one embodiment of the composition, the linker peptides are 1-20 aminoacids long. A non-limiting example of PA fragment capable of binding LFnor EFn is PA63. In one embodiment of a composition described hereinwhich includes a PA protein, the PA protein is an oligomeric PA. Inanother embodiment, the PA is a native anthrax toxin protective antigen(PA) protein. In another embodiment, the oligomeric PA is bound to thefusion protein.

In one embodiment, compositions described herein which include toxinsand/or toxin fusions are useful for the treatment of pain such as nerve,joint, skin, visceral, bladder, or muscle pain, diabetic neuropathicpain, cancer pain, fibromyalgia or other systemic pain disorders. Inanother embodiment, these compositions are useful in the manufacture ofmedicament for the treatment of pain.

AB Toxin—PAd4/PA Fusion Proteins

In another aspect, provided herein is a fusion protein comprising an ABtoxin fused to a linker peptide that is then linked to a PAd4 domain ofPA, wherein PAd4 is the C-terminal receptor-binding domain of anthraxtoxin protective antigen, wherein the fusion protein further comprises atranslocation domain, a holotoxin or a mutant form of the holotoxin thathas been modified (e.g., chemically) or mutated to negate the toxinreceptor-binding function of the AB toxin. In one embodiment of a fusionprotein, PA is used instead of a PAd4 domain. In one embodiment, the PAis a variant PA mutant form that is resistant to cleavage by furin,(PA^(furin-)).

In one embodiment, provided herein is a composition comprising an ABtoxin fused to a linker peptide operably linked to a PAd4 domain of PA,wherein Pad4 is the C-terminal receptor-binding domain of anthrax toxinprotective antigen, wherein the fusion protein further comprises atranslocation domain, a holotoxin or a mutant form of the holotoxin thathas been modified (e.g., chemically) or mutated to negate the toxinreceptor-binding function of the AB toxin. In one embodiment of a fusionprotein, PA is used instead of a PAd4 domain. In one embodiment, the PAis a variant PA mutant form that is resistant to cleavage by furin,(PA^(furin-)). In some embodiments of all the aspects described herein,a composition as described herein can further comprise apharmaceutically acceptable carrier or excipient.

In one aspect, provided herein is a fusion protein comprising: (a) an ABtoxin; (b) an anthrax toxin protective antigen (PA) or a PA fragmentthereof, wherein the PA or fragment binds a receptor expressed on thenociceptor neuron; and (c) a translocation domain (TL) that is capableof translocating the protease from within an endosome, across theendosomal membrane and into the cytosol of the nociceptor neuron.

In one embodiment, provided herein is a composition comprising a fusionprotein comprising: (a) an AB toxin; (b) an anthrax toxin protectiveantigen (PA) or a PA fragment thereof, wherein the PA or fragment bindsa receptor expressed on the nociceptor neuron; and (c) a translocationdomain (TL) that is capable of translocating the toxin, a protease, fromwithin an endosome, across the endosomal membrane and into the cytosolof the nociceptor neuron. In some embodiments of all the aspectsdescribed herein, a composition as described herein can further comprisea pharmaceutically acceptable carrier or excipient.

In one embodiment of a fusion protein or composition described herein,the AB toxin is selected from Ricin toxin, Cholera toxin A-part andB-part; Pseudomonas aeruginosa Exotoxin A A-part and B-part; Shiga toxinA-part and B-part; and Diphtheria toxin A-part and B-part.

In one embodiment of a fusion protein or composition described herein,the PA-binding receptor expressed on the nociceptor neuron is ANTXR2(CMG2).

In one embodiment of a fusion protein or composition described herein,the PA fragment is a C-terminal receptor-binding domain of PA, e.g.,PA63, PAd2 and PAd4, or PAd4 as non-limiting examples.

In one embodiment of a fusion protein or composition described herein,the C-terminal receptor-binding domain of PA comprises, consistessentially of, or consists of a domain having the amino acid sequenceof SEQ ID NO: 35-40. In one embodiment of a fusion protein orcomposition described herein, the PA fragment comprises, consistessentially of, or consists of a domain having the amino acid sequenceof SEQ ID NO: 35-40.

In one embodiment of a fusion protein or a composition described herein,the C-terminal receptor-binding domain of PA comprises the PAd2 that isinvolved in membrane insertion and heptamerization. In one embodiment,PAd2 is located at residues 259-487 of PA (SEQ. ID. NO:1). In oneembodiment of a fusion protein or a composition described herein, theC-terminal receptor-binding domain of PA comprises the PAd4 that isinvolved in host cell receptor binding. In one embodiment, PAd4 islocated at residues 595-735 of PA (SEQ. ID. NO:1).

In one embodiment of a fusion protein or a composition described herein,the C-terminal receptor-binding domain of PA comprises, consists of, orconsist essentially of the PAd2 and the PAd4domain of PA. For example,the C-terminal receptor-binding domain of PA comprises, consists of, orconsist essentially of the residues 259-487 and 488-735 of PA (SEQ. ID.NO:1). Alternately, the C-terminal receptor-binding domain of PAcomprises, consists of, or consist essentially of the residues 259-487and 488-764 of PA (SEQ. ID. NO:1).

In one embodiment of a fusion protein or composition described herein,the PAd4 or PA fragment is linked with the AB toxin with a peptidelinker. In one embodiment, the linker peptide is 1-20 amino acids long.In one embodiment of a fusion protein or composition described,approximately about 1-60 consecutive amino acids from the N-terminalside adjacent to the native PAd4 domain are further incorporated betweenthe AB toxin and the PAd4. In one embodiment of a fusion protein orcomposition, the PAd4 is located at the C-terminus of the fusionprotein, and the AB toxin is at the C-terminus of the fusion protein. Inone embodiment of a fusion protein or composition, the PAd4 is locatedat the N-terminus of the fusion protein. In another embodiment of afusion protein or composition, the PAd4 is located both at theN-terminus and the C-terminus of the fusion protein, with the AB toxinsandwiched between the two PAd4 domains. In another embodiment of afusion protein or composition, there are more than one PAd4 domain inthe fusion protein, e.g., 2-10 PAd4 domains, 1-5 PAd4 domains, 2-5 PAd4domains, in tandem. In one embodiment of a fusion protein or compositiondescribed herein, where multiple PAd4 domains are present, the multiplePAd4 domains are arranged in tandem. In one embodiment of a fusionprotein or composition described herein, the multiple PAd4 domains arelinked by peptide linkers. In one embodiment of a fusion protein orcomposition described herein, the TL, translocation domain; a holotoxin;or a mutant form of the holotoxin is sandwiched between the AB toxin andPAd4 or PA fragment.

In one embodiment of a fusion protein or composition described herein,the AB toxin is selected from Ricin toxin, Cholera toxin A-part andB-part; Pseudomonas aeruginosa Exotoxin A A-part and B-part; Shiga toxinA-part and B-part; and Diphtheria toxin A-part and B-part. In oneembodiment of a fusion protein or composition described herein, the TLis a clostridial neurotoxin translocation domain; a holotoxin; or amutant form of the holotoxin that have been modified (e.g., chemically)or mutated to negate the toxin receptor-binding function of the ABtoxin. In some embodiments of a fusion protein or composition describedherein, the translocation domain is derived from BTx or TTx, e.g., theH_(N) of BTx or TTx as disclosed in Table 1 (a clostridial neurotoxintranslocation domain), or the translocation domain is derived from theanthrax toxin, e.g., LFn or EFn, or a polycationic sequence describedherein. In some embodiments of the fusion proteins or compositionsdescribed herein, the holotoxin or a mutant form of the holotoxin isselected from Ricin toxin, Cholera toxin, PE; Shiga toxin, DT; andscorpion long-or short-chain toxins. In some embodiments of a fusionprotein or composition described herein, the holotoxin or a mutant formof the holotoxin is PA, mPA, or PA^(furin-), e.g., for an entire PAprotein, the furin cleavage site comprising amino acid residues RKKR(residues 164-167 of SEQ ID NO: 1 minus the 29 aa signal peptide in SEQID NO:1) has been replaced by a furin-resistant amino acid sequence suchas SSSR (SEQ ID NO: 32) or SSSS (SEQ ID NO: 33).

In some embodiments of a fusion protein described herein comprising aLFn or EFn, or LF or EF, these fusion proteins can be used together withnon-fused or separate PA, or used together with a second fusion proteincomprising (a) PA or PA fragment capable of binding LFn or EFn, and (b)a nociceptor neuron-binding protein, where the PA or PA fragment isfused to a nociceptor neuron-binding protein, and the nociceptorneuron-binding protein directs the toxin to nociceptor neurons to treatpain. In other words, in one embodiment, the fusion protein describedherein comprising a LFn or EFn, or LF or EF is co-administered with PAalone to a subject to treat pain. In another embodiment, the fusionprotein described herein comprising an LFn or EFn, or LF or EF isco-administered with a second fusion protein to a subject to treat pain.In such instances, the second fusion protein comprises (a) PA or PAfragment capable of binding LFn or EFn, and (b) a nociceptorneuron-binding protein, where the PA or PA fragment is fused to anociceptor neuron-binding protein, and the nociceptor neuron-bindingprotein directs the first fusion protein containing the toxin tonociceptor neurons to treat pain. In some embodiments of the secondfusion protein, the PA is a variant, modified (e.g., chemically) ormutated form (mPA) that does not bind the ANTXR2 receptor as describedherein.

In one aspect, provided herein is a composition comprising:

(I) a first fusion protein comprising (a) AB toxin and (b)(i) theN-terminal domain (LFn) of anthrax toxin lethal factor or (b)(ii) theN-terminal domain (EFn) of anthrax toxin edema factor, and

(II) a second protein comprising (c) PA or PA fragment capable ofbinding LFn or EFn, and optionally (d) nociceptor neuron-bindingprotein, wherein the parts (a) and (b) are joined by a peptide linker,and wherein the parts (c) and (d) are also joined by a peptide linker,when part (d) is present. In one embodiment of the compositiondescribed, when the second protein is a fusion protein, the PA is amutant variant of PA, an mPA.

In one embodiment of the composition, the linker peptides are 1-20 aminoacids long. An example of PA fragment capable of binding LFn or EFn isPA63. In one embodiment of any one composition described herein, the PAprotein is an oligomeric PA. In one embodiment of any one compositiondescribed herein, the PA is a native anthrax toxin protective antigen(PA) protein. In one embodiment, the PA is the oligomeric PA. In oneembodiment, the oligomeric PA is bound to the fusion protein. In oneembodiment, this composition is useful for the treatment of pain such asnerve, joint, skin, visceral, bladder, or muscle pain, and diabeticneuropathic pain, cancer pain, fibromyalgia or other systemic paindisorders. In another embodiment, this composition is useful in themanufacture of medicament for the treatment of pain.

ICK Toxin—PAd4/PA Fusion Proteins

Also described herein is the engineering of anthrax toxin componentswith an inhibitor cysteine knot (ICK) toxin (e.g., a Conotoxin (CTx)).Because some ICK toxins are known to modulate the activity of ionchannels, they have been used to treat pain but effects on cell-typesother than nociceptors have hampered systemic treatment. If these toxinsare fused to, or otherwise used to decorate, PAd4 or native PA, they canbe targeted specifically to nociceptors. (See FIG. 9) Accordingly, inone aspect, an engineered fusion protein comprises (a) PAd4 or native PAor receptor-binding fragment thereof, and (b) an ICK toxin. In anotherembodiment, provided herein is a composition comprising an engineeredfusion protein that comprises (a) PAd4 or native PA or receptor-bindingfragment thereof, and (b) an ICK toxin. In some embodiments of all theaspects described herein, a composition as described herein can furthercomprise a pharmaceutically acceptable carrier or excipient. The PAd4 ornative PA or receptor-binding fragment thereof can be fused to the ICKtoxin by a peptide linker. In one embodiment, the linker peptide is 1-20amino acids long.

ICK Toxin—LFn/EFn Fusion Proteins

Alternatively, the ICK toxin can be fused to LFn or EFn, or a fusionprotein containing LFn or EFn, which can then be used in combinationwith PA, or a modified (e.g., chemically) or mutated form of PA, toaffect nociceptors specifically. Accordingly, in one aspect, providedherein is an engineered fusion protein comprising (a) LFn and (b) an ICKtoxin. In another aspect, provided herein is an engineered fusionprotein comprising (a) EFn, and (b) an ICK toxin. In another aspect,provided herein is an engineered fusion protein comprising (a) an ICKtoxin, and (b) a nociceptor neuron-binding protein. In one embodiment,provided herein is a composition comprising an engineered fusion proteincomprising (a) LFn and (b) an ICK toxin. In another embodiment, providedherein is a composition comprising an engineered fusion proteincomprising (a) EFn, and (b) an ICK toxin. In another embodiment,provided herein is a composition comprising an engineered fusion proteincomprising (a) an ICK toxin, and (b) a nociceptor neuron-bindingprotein. The nociceptor neuron-binding protein helps direct the ICKtoxin specifically to the nociceptor neuron. Similarly, the LFn or EFn,together with PA or variant forms of PA or receptor-binding fragmentsthereof, helps deliver the toxin directly to nociceptor neurons and intothe cytosol. In one embodiment of an engineered fusion protein orcomposition described herein, the LFn, EFn, or nociceptor neuron-bindingprotein is fused to the ICK toxin by a peptide linker. In oneembodiment, the linker peptide is 1-20 amino acids long. In oneembodiment of an engineered fusion protein or composition describedherein, the LFn, EFn, or nociceptor neuron-binding protein is located atthe N-terminus of the fusion protein. In one embodiment of an engineeredfusion protein or composition described herein, the LFn, EFn, ornociceptor neuron-binding protein is located at the C-terminus of thefusion protein. In another embodiment, the LFn, EFn, or nociceptorneuron-binding protein is located at both the N-terminus and theC-terminus of the fusion protein, with the ICK toxin sandwiched betweenthe LFn, EFn, or nociceptor neuron-binding protein.

Disulfide-Containing Peptide Toxin—LFn/EFn Fusion Proteins

In another aspect, provided herein is a fusion protein comprising (a) adisulfide-containing peptide toxin and (b)(i) the N-terminal domain(LFn) of anthrax toxin lethal factor or (b)(ii) the N-terminal domain(EFn) of anthrax toxin edema factor. In one embodiment, a composition isprovided comprising a fusion protein comprising (a) adisulfide-containing peptide toxin and (b)(i) the N-terminal domain(LFn) of anthrax toxin lethal factor or (b)(ii) the N-terminal domain(EFn) of anthrax toxin edema factor. In one embodiment of all theaspects described herein, a composition as described herein can furthercomprise a pharmaceutically acceptable carrier or excipient.

In one embodiment of a fusion protein or composition described, thedisulfide-containing peptide toxin is linked N-terminally orC-terminally or both N-terminally and C-terminally, or chemicallycrosslinked at one or more sites to LFn or EFn. In one embodiment of afusion protein or composition described herein, the disulfide-containingpeptide toxin is linked to LFn or EFn. LFn is a domain of anthrax toxinlethal factor which binds to oligomeric forms of PA63, theproteolytically activated form of anthrax PA. EFn is a domain of anthraxtoxin edema factor, which domain binds to oligomeric forms of PA63. Inone embodiment of a fusion protein or composition described herein, thedisulfide-containing peptide toxin is an inhibitor cysteine knot toxin(ICK) toxin. In one embodiment of a fusion protein or compositiondescribed herein, the disulfide-containing peptide toxin is a conotoxin,an agatoxin, a delta-palutoxin, a huwentotoxin or a ProTx II toxin. Inone embodiment of a fusion protein or composition, part (a) is fusedwith part (b) with a linker peptide. In one embodiment, the linkerpeptide is 1-20 amino acids long. In one embodiment of a fusion proteinor composition described herein, the LFn or EFn is located at theN-terminus of the fusion protein. In one embodiment of a fusion proteinor composition described herein, the LFn or EFn is located at theC-terminus of the fusion protein. In another embodiment of a fusionprotein or composition, the LFn or EFn is located at both the N-terminusand the C-terminus of the fusion protein, with the disulfide-containingpeptide toxin sandwiched between the two LFns or EFns.

In one aspect, described herein is a fusion protein comprising: (a) adisulfide-containing peptide toxin and (b) a protein capable of bindingto an anthrax toxin protective antigen (PA) or a fragment thereof,wherein the fragment binds a receptor expressed on a nociceptor neuron.In one embodiment, the disulfide-containing peptide toxin is a channelblocking toxin having a cysteine-knot motif that is capable of blockingsodium or calcium or both sodium and calcium channels in a nociceptorneuron. In one embodiment, a composition is provided comprising a fusionprotein comprising: (a) a disulfide-containing peptide toxin and (b) aprotein capable of binding to an anthrax toxin protective antigen (PA)or a fragment thereof, wherein the PA fragment binds a receptorexpressed on the nociceptor neuron. In some embodiments of all theaspects described herein, a composition as described herein can furthercomprise a pharmaceutically acceptable carrier or excipient.

In all of the above aspects and embodiments of a fusion protein orcomposition described, the disulfide-containing peptide toxin cancomprise a cysteine knot motif.

In all of the above aspects and embodiments of a fusion protein orcomposition described, the disulfide-containing peptide toxin can be aconotoxin, an agatoxin, a delta-palutoxin, a huwentotoxin or a ProTx IItoxin.

In all of the above aspects and embodiments of a fusion protein orcomposition described, the PA-binding receptor expressed on thenociceptor neuron can be ANTXR2 (CMG2).

In all of the above aspects and embodiments of a fusion protein orcomposition described, the protein capable of binding to PA can be: (i)an anthrax toxin lethal factor (LF); or (ii) an anthrax toxin edemafactor (EF).

In all of the above aspects and embodiments of a fusion protein orcomposition described, the PA binding domain of LF is the N-terminaldomain of LF, (abbreviated as LFPABD or LFn).

In all of the above aspects and embodiments of a fusion protein orcomposition described, the PA binding domain of EF is the N-terminaldomain of EF, (abbreviated as EFPABD or EFn).

In some embodiments of a fusion protein described herein comprising aLFn or EFn, or LF or EF, these fusion proteins are used or administeredtogether with unfused PA, or used or administered together with a secondfusion protein comprising (a) PA or PA fragment capable of binding LFnor EFn, and (b) a nociceptor neuron-binding protein, where the PA or PAfragment is fused to a nociceptor neuron-binding protein, and thenociceptor neuron-binding protein directs the toxin to nociceptorneurons to treat pain. In other words, in one embodiment, the fusionprotein described herein comprising an LFn or EFn, or LF or EF isco-administered with unfused or native PA to a subject to treat pain. Inanother embodiment, the fusion protein described herein comprising anLFn or EFn, or LF or EF is co-administered with a second fusion proteinto a subject to treat pain. The second fusion protein comprises (a) PAor PA fragment capable of binding LFn or EFn, and (b) a nociceptorneuron-binding protein, where the PA or PA fragment is fused to anociceptor neuron-binding protein, and the nociceptor neuron-bindingprotein directs the first fusion protein containing the toxin tonociceptor neurons to treat pain. In some embodiments of the secondfusion protein, the PA is a modified (e.g., chemically) or mutatedvariant form (mPA), that does not bind the ANTXR2 receptor as describedherein.

In one aspect, described herein is a composition comprising:

(I) a first fusion protein comprising (a) a disulfide-containing peptidetoxin and (b)(i) the N-terminal domain (LFn) of anthrax toxin lethalfactor or (b)(ii) the N-terminal domain (EFn) of anthrax toxin edemafactor, and

(II) a second protein comprising (c) PA or PA fragment capable ofbinding LFn or EFn, and optionally (d) nociceptor neuron-bindingprotein, wherein the parts (a) and (b) are joined by a peptide linker,and wherein the parts (c) and (d) are also joined by a peptide linker.In one embodiment of the composition described, when the second proteinis a fusion protein, the PA is a mutant variant of PA, an mPA. In oneembodiment of all the aspects described herein, a composition asdescribed herein can further comprise a pharmaceutically acceptablecarrier or excipient.

In one embodiment, the linker peptides are 1-20 amino acids long. Anon-limiting example of a PA fragment capable of binding LFn or EFn isPA63. In one embodiment of a composition described herein, the PAprotein is an oligomeric PA. In one embodiment of a compositiondescribed herein, the PA is a native anthrax toxin protective antigen(PA) protein. In one embodiment of a composition described herein, thePA is an oligomeric PA, which can be bound to the fusion protein. In oneembodiment, such a composition is useful for the treatment of pain suchas nerve, joint, skin, visceral, bladder, or muscle pain, and diabeticneuropathic pain, cancer pain, fibromyalgia or other systemic paindisorders. In another embodiment, this composition is useful in themanufacture of medicament for the treatment of pain.

Disulfide-Containing Peptide Toxin—PAd4/PA Fusion Proteins

In one aspect, provided herein is a fusion protein comprising (a) adisulfide-containing peptide toxin, and (b)(i) an anthrax toxinprotective antigen (PA) or (b)(ii) an anthrax toxin protective antigenC-terminal receptor binding domain (PAd4); or (b)(iii) a nociceptorneuron-binding protein. (See FIG. 9) In one embodiment, a composition isprovided comprising a fusion protein comprising (a) adisulfide-containing peptide toxin, and (b)(i) an anthrax toxinprotective antigen (PA) or (b)(ii) an anthrax toxin protective antigenC-terminal receptor binding domain (PAd4); or (b)(iii) a nociceptorneuron-binding protein. In one embodiment of a fusion protein orcomposition, part (a) is fused to part (b) with a linker peptide. In oneembodiment, the linker peptide is 1-20 amino acids long. In oneembodiment of a fusion protein or composition described herein, thedisulfide-containing peptide toxin is located at the N-terminus of thefusion protein. In one embodiment of a fusion protein or compositiondescribed herein, the disulfide-containing peptide toxin is located atthe C-terminus of the fusion protein. In another embodiment, the PA,PAd4 or nociceptor neuron-binding protein is located at both theN-terminus and the C-terminus of the fusion protein, such that thedisulfide-containing peptide toxin is sandwiched by the two PA, PAd4 ornociceptor neuron-binding proteins. In one embodiment of a fusionprotein or composition described herein, the disulfide-containingpeptide toxin is an inhibitor cysteine knot toxin (ICK) toxin. In oneembodiment of a fusion protein or composition described herein, thedisulfide-containing peptide toxin is a conotoxin, an agatoxin, adelta-palutoxin, a huwentotoxin or a ProTx II (PE) toxin. In oneembodiment of a fusion protein or composition described herein, thefusion protein comprises a linker peptide between the PA, PAd4 ornociceptor-binding protein and the inhibitor cysteine knot toxin.

In another aspect, provided herein is a fusion protein comprising: (a) adisulfide-containing peptide toxin that is capable of blocking sodium orcalcium or both sodium and calcium channels in a nociceptor neuron; and(b) a targeting moiety (TM) that is capable of binding to a binding siteon the nociceptor neuron, wherein the nociceptor neuron expresses sodiumor calcium or both sodium and calcium channels. In one embodiment, thedisulfide-containing peptide toxin is a channel blocking toxin having acysteine-knot motif that is capable of blocking sodium or calcium orboth sodium and calcium channels in a nociceptor neuron. In anotherembodiment, provided herein is a composition comprising a fusion proteincomprising: (a) a disulfide-containing peptide toxin that is capable ofblocking sodium or calcium or both sodium and calcium channels in anociceptor neuron; and (b) a targeting moiety (TM) that is capable ofbinding to a binding site on the nociceptor neuron, wherein thenociceptor neuron expresses the sodium or calcium or both sodium andcalcium channels therein. In some embodiments of all the aspectsdescribed herein, a composition as described herein can further comprisea pharmaceutically acceptable carrier or excipient.

In all of the above aspects and embodiments of a fusion protein orcomposition described, the disulfide-containing peptide toxin cancomprises a cysteine knot motif.

In all of the above aspects and embodiments of a fusion protein orcomposition described, the disulfide-containing peptide toxin can be,for example, a conotoxin, an agatoxin, a delta-palutoxin, a huwentotoxinor a ProTx II toxin.

In all of the above aspects and embodiments of a fusion protein orcomposition described, the TM can be selected, for example, from thegroup consisting of: (i) an anthrax toxin protective antigen (PA); (ii)a C-terminal receptor-binding domain of PA; or (iii) a PA fragmentthereof, e.g., PAd4; or (iv) a nociceptor neuron-binding protein.

In one embodiment of each of the fusion protein or compositiondescribed, the TM, PA or C-terminal receptor-binding domain of PA, or aPA fragment or the nociceptor neuron-binding protein can bind the ANTXR2(CMG2) receptor expressed on the nociceptor neuron. In one embodiment,the nociceptor neuron-binding protein is an antibody that specificallybinds the ANTXR2 receptor. In one embodiment, the antibody is anantibody fragment that can bind the ANTXR2 receptor. In anotherembodiment, the antibody specifically binds to the NGF receptor, or anion-channel protein present on nociceptor neurons. In some embodiments,the ion-channel protein is selected from Nav1.7, Nav1.8 or Nav1.9.

In all of the above aspects and embodiments of a fusion protein orcomposition described, the PA can be, for example, a mutant PA resistantto furin cleavage as described herein and known in the art. For example,the furin cleavage site comprising amino acid residues RKKR can bereplaced by a furin-resistant amino acid sequence such as SSSR (SEQ IDNO: 32) or SSSS (SEQ ID NO: 33). RKKR are the residues 164-167 of SEQ IDNO: 1 minus the 29 amino acid signal peptide in SEQ ID NO:1.

In all of the above aspects and embodiments of a fusion protein orcomposition described, the C-terminal receptor-binding domain of PA canbe, for example, PA63 or PAd4.

In all of the above aspects and embodiments of a fusion protein orcomposition described, the PAd4, the PA or the C-terminal receptorbinding domain of PA can be modified or mutated, for example, to beresistant to cleavage by a protease. For example, one or more, up to andincluding each of the Lys residues at positions 594, 613, 633, 637, 653,673, 679, 680, 684, 695, 703, 722, 723, 729, and 730 of SEQ ID NO: 1(minus the 29 aa signal peptide in SEQ. ID. NO:1) can be replaced, forexample, by Arg or His. In other words, one or more, up to and includingeach of the Lys residues in the PAd4 domain of the PA at positions 623,642, 662, 666, 682, 702, 708, 709, 713, 724, 732, 751, 752, 758, and 759in SEQ. ID. NO:1 can be replaced, for example, by Arg or His. Otherexamples of proteases which the PAd4, the PA or the C-terminal receptorbinding domain of PA can be made resistant to but are not limited to arelysyl peptidase, trypsin, Enterokinase, clostripain, elastase,chymotrypsin, thermolysin, endoproteinase Lys-C, and endoproteinaseArg-C.

(mPA—Nociceptor-Binding Protein) Fusion Protein

Further described herein is the engineering of mPA, a variant modified(e.g., chemically) or mutated PA, so as to block its nativereceptor-binding function, as described, e.g., in U.S. PatentApplication Publication No. 20150044210, incorporated herein byreference in its entirety) to be fused with molecules that can targetnociceptor surface receptors or ion channels specifically. Accordingly,in one aspect, the engineered fusion protein comprises (a) anengineering mPA, and (b) a nociceptor-binding protein. In oneembodiment, provided herein is a composition comprising a engineeredfusion protein comprises (a) an engineering mPA, and (b) anociceptor-binding protein. As examples, the nociceptor-binding proteincan be a non-PA protein capable of binding the ANTRAX receptor on thenociceptor, or an antibody that binds a receptor or ion channel proteinson the cell surface of the nociceptor, or a protein ligand of a receptoron the cell surface of the nociceptor. As examples of the describedengineered fusion protein, the mPA can be fused to NGF which targets andbinds the NGF receptor, or mPA can be fused to antibodies or antibodyfragments that specifically bind the Nav1.7 channel which creates sodiumion pores on nociceptors. Nav1.7 is usually expressed at high levels intwo types of neurons, the nociceptive (pain) neurons at dorsal rootganglion (DRG) and trigeminal ganglion, and sympathetic ganglionneurons, which are part of the autonomic (involuntary) nervous system.When such a fusion protein comprising an mPA fused to nociceptor-bindingprotein is used in conjunction with another fusion protein comprisingLFn or EFn or EF or LF, e.g., LFn fused to BTx, or LFn fused to TTx, orEFn fused to BTx, or EFn fused to the TTx, and when the mPA/LFn or EFninteraction occurs between the first fusion protein and the secondfusion protein, the toxins are specifically directed to nociceptors bythe nociceptor-binding protein of the fusion protein, and the toxinenters the cytosol by way of the mPA/LFn or EFn interaction.

Also provided herein are native PA or mutant PA (mPA, which denotes avariant PA modified (e.g., chemically) or mutated, that has its nativereceptor-binding function blocked), fused with molecules that can targetnociceptor surface molecules in combination with LF or EF. In someembodiments, the fusion protein comprising an mPA fused to anociceptor-binding protein is used specifically in conjunction with LFor EF. MAP kinases and their signaling pathways have been shown to beimportant for chronic pain development in mouse models. LF specificallyinhibits MAP kinase signaling. In one embodiment, PA or mPA incombination with LF can be used to specifically target MAP kinasesignaling in nociceptors to block pain. EF activates adenylate cyclase,which has also been linked to pain development. One can also targetadenylate cyclase in pain by using PA or mPA in combination with EF.

Nav1.8 and Nav1.9 can also be used as target receptors for thenociceptor-binding protein of a fusion protein as described herein. Inother embodiments of the engineered fusion protein described herein, thenociceptor-binding protein part of the fusion protein binds Nav1.8 orNav1.9.

In any of the above aspects and embodiments of a fusion protein orcomposition described herein comprising an LFn or EFn, or LF or EF, andembodiments of a fusion protein or composition described hereincomprising PA or PA fragments, an oligomeric form of PA formed fromproteolytically activated PA (e.g., PA63) or mPA can be substituted formonomeric PA to increase avidity for receptor-bearing cells. The toxineffector moiety can be bound to the PA oligomeric form beforeadministering, or injected separately.

In another aspect, described herein is an engineered fusion proteincomprising:

-   a first domain comprising a polypeptide selected from the group    consisting of:    -   i) an anthrax toxin protective antigen (PA) moiety; or    -   ii) a mutant anthrax toxin protective antigen (mPA) moiety that        has been altered to block its native receptor-binding function,        fused with a molecule capable of specifically targeting a        nociceptor surface receptor or an ion channel receptor; and-   a second domain comprising a polypeptide selected from the group    consisting of:    -   iii) an inhibitor cysteine knot (ICK) toxin (e.g., a Conotoxin        (CTx)) optionally fused with an anthrax toxin translocation        peptide;    -   iv) an intracellularly acting toxin catalytic domain optionally        fused with an anthrax toxin translocation peptide; or    -   v) an anthrax toxin edema factor (EF) and/or anthrax toxin        lethal factor (LF).

In one embodiment, the first domain and the second domain are fusedtogether with a linker peptide. In one embodiment, the linker peptide is1-20 amino acids long. In another embodiment, the linker peptide isstable in human serum for at least 1 minute.

In another embodiment, the first domain of the fusion protein serves asthe targeting moiety (TM) of the fusion protein. The targeting moietyfunctions to direct the toxin to the nociceptor neuron via a cellsurface marker enriched on the nociceptor. Non-limiting examplesinclude, but are not limited to the abundant ANTXR2 receptor, NGFreceptor, and/or the Nav1.7, Nav1.8, or Nav1.9 ion channels expressed onthe surface of nociceptors. In one embodiment, the targeting moiety isone that is capable of binding to a binding site on the nociceptorneuron, which binding site is capable of undergoing endocytosis to beincorporated into an endosome within the nociceptor neuron, and whereinthe nociceptor neuron expresses a SNARE protein which is subsequentlyproteolytically cleaved by the toxin of the second domain. In anotherembodiment, the targeting moiety is one that is capable of binding to abinding site on the nociceptor neuron, wherein the nociceptor neuronexpresses sodium or calcium, or both sodium and calcium, ion channels(e.g., Nav1.7, Nav1.8, and Nav1.9 ion channels), or expresses ANTXR2receptor or NGF receptor. In one embodiment, the binding site on thenociceptor neuron is, accordingly, ANTXR2 receptor, NGF receptor,Nav1.7, Nav1.8, or Nav1.9 ion channel.

The molecule capable of specifically targeting a nociceptor surfacereceptor, such as ANTXR2, or an ion channel receptor can be, e.g. apolynucleotide (e.g. an aptamer), a polypeptide, an antibody orantigen-binding fragment thereof, or an affibody. Affibody molecules aresmall proteins engineered to bind to a large number of target proteinsor peptides with high affinity, imitating monoclonal antibodies, and aretherefore a member of the family of antibody mimetics. In someembodiments, the molecule can be an antibody reagent, e.g. an antibody,monoclonal antibody, and/or antigen-binding portion thereof. Nociceptorsurface receptors and/or nociceptor ion channel receptors are known inthe art and are described in, e.g., Gohar. Modulator 2005 19:9-13;Bennaroch. Neurology 2015 10; Simon et al. The Nociceptive Membrane.Academic Press 2011; each of which is incorporated by reference hereinin its entirety. Non-limiting examples of nociceptor surface receptorsand/or nociceptor ion channel receptors include, e.g., NGF receptor(NGFR) (e.g. NCBI Gene ID: 4804) Nav1.7 (e.g. NCBI Gene ID: 6335),Nav1.8 (e.g. NCBI Gene ID: 6336), or Nav1.9 (e.g. NCBI Gene ID: 11280).All database sequences as referred herein and throughout thespecification by sequence reference numbers are incorporated herein byreference in their entirety. The database reference numbers andsequences are as set forth in the databases on the filing date of thisapplication. In one embodiment of any aspect involving a moleculecapable of specifically targeting a nociceptor surface receptor, themolecule can be an antibody reagent that specifically binds to the NGFreceptor and/or an antibody reagent that specifically binds to Nav1.7,Nav1.8 or Nav1.9. In some embodiments of any such aspect, in order todecrease off-target effects, multiple molecules capable of specificallytargeting a nociceptor surface receptor or an ion channel receptor canbe present in the same fusion protein and/or composition. Thecomposition may also comprise two, three or more fusion proteins withdifferent targeting moieties.

In one embodiment of any of the aspects described herein that involve aPA polypeptide, the pore-forming ability of the PA is desired to betargeted without using PA's ability to specifically bind ANTXR2.Accordingly, in one embodiment of any aspect involving a PA polypeptide,the composition can comprise a mutant anthrax toxin protective antigen(mPA) moiety that has been altered to block its native ANTXR2-bindingfunction, fused with a molecule capable of specifically targeting anociceptor surface receptor or an ion channel receptor. By way ofnon-limiting example, the PAd4 domain of PA can be deleted and/oraltered to block the receptor-binding function. By way of furthernon-limiting example, mPA can comprise N682A and/or D683A (relative tothe sequence of SEQ ID NO: 1 after the removal of the signal peptides ataa 1-29 residues) (see, e.g., Rosovitz M J, et al. 2003.Alanine-scanning mutations in domain 4 of anthrax toxin protectiveantigen (PAd4) reveal residues important for binding to cellularreceptor and to a neutralizing monoclonal antibody. J. Biol. Chem.278:30936-30944; which is incorporated by reference herein in itsentirety). Various methods of altering polypeptide sequences and/orengineering proteins to comprise desired mutations are known in the art,and examples of them are described elsewhere herein.

In one embodiment of an engineered fusion protein described herein, thesecond domain of the fusion protein is the toxin effector moiety of thefusion protein. Depending upon the toxin protein, the toxin effectormoiety functions to disrupt cell signaling on the nociceptors and/orblocks the release of neurotransmitters from the nociceptors or killsthe nociceptors. The toxin effector moiety can be a non-cytotoxicprotease that targets one or more SNARE proteins on vesicles (e.g., BTxand TTx), MAP kinases (EF and LF), etc.

As described herein, the inventors have discovered that the anthraxprotective antigen can selectively target nociceptor neurons, binding tothem and forming a pore capable of membrane transport. By coupling(either physically or functionally), an anthrax toxin protective antigenand an appropriate toxin, the nociceptor neurons can be killed and/ordisabled or the pain signal can be blocked. Such compositions can beused, e.g. to treat pain, e.g., by disabling cell signaling or synaptictransmission in nociceptor neurons without substantial off-targeteffects on the rest of the nervous system. Moreover, use of this systemallows a pain treatment without the debilitating side effect ofsubstance abuse.

In certain aspects, the fusion proteins described herein can comprise atoxin. As used herein, “toxin” refers to a compound produced by anorganism which causes or initiates the development of a noxious,poisonous or deleterious effect in a host cell presented with the toxin.Such deleterious conditions may include inhibition of key cellularfunctions, inhibition of cell metabolism, and/or cell death.

In some embodiments of all the aspects described herein involving atoxin, the toxin can be an inhibitor cysteine knot (ICK) toxin, e.g., aconotoxin (CTx).

In some embodiments of all the aspects described herein involving atoxin, the toxin can be an intracellularly acting toxin and/or anintracellularly acting toxin catalytic domain. Suitable bacterial toxinscan include those with proteolytic activity against, e.g. SNARE proteinsto prevent neurotransmitter release, or toxins that are cytotoxic toneurons. Non-limiting examples of suitable toxins can include bacterialtoxins such as, e.g., diphtheria toxin (DTx) (e.g. NCBI Gene ID:2650491; SEQ ID NO: 2); Pseudomonas aeruginosa exotoxin A (PTx or PE)(e.g. NCBI Gene ID: 877850; SEQ ID NO: 3); botulinum toxin (BTx) (e.g.NCBI Gene ID: 5398487; SEQ ID NO: 4); tetanus toxin (TTx) (e.g. NCBIGene ID: 17583237; SEQ ID NO: 5) shiga toxin (e.g. Shigella Stx (e.g.,GenBank accession numbers CAC05622 and CAC05623) Stx-1 (e.g. GenBankaccession numbers 32400300 and 32400299) and/or Stx-2 (e.g. GenBankaccession numbers 161511882 and 161511883), anthrax lethal toxin (lethalfactor), and/or anthrax edema toxin (edema factor). A furthernon-limiting example of a suitable toxin is ricin toxin (e.g. NCBI GeneID: 8287993).

In some embodiments of all the aspects described herein, the toxin canbe an anthrax toxin edema factor (EF) and/or anthrax toxin lethal factor(LF).

In some embodiments of all the aspects described herein, multiple toxinscan be present in the same fusion protein and/or composition asdescribed herein.

In some embodiments, an effector moiety includes a toxin that comprisesa translocation domain (TL) therein. In one embodiment, the TL iscapable of translocating the fusion protein from within an endosome,across the endosomal membrane and into the cytosol of a nociceptorneuron. In some embodiments, the TL is the anthrax translocation signalpeptide (LFn or EFn), also referred to herein as the anthrax toxintranslocation peptide, a H_(N) domain of BTx (serotypes included) or theH_(N) domain of TTx, or a polycationic sequence such as KKK, KKKKKK (SEQID NO: 59), KKKKKKKK (SEQ ID NO: 60), HHH, HHHHHH (SEQ ID NO: 61),HHHHHHHH (SEQ ID NO: 62), RRR, RRRRRR (SEQ ID NO: 63), or RRRRRRRR (SEQID NO: 64). See US Patent Application Publication No: US 2003/0202989,which is incorporated here by reference in its entirety. In otherembodiments, the TL is a clostridial neurotoxin translocation domain,H_(N), derived from a clostridial neurotoxin (CNT) family memberprotein. This includes the BTx serotypes, TTx and the newly discoverednon-Clostridial botulinum-like toxin.

In some embodiments of all the aspects described herein, the toxin canbe fused with an anthrax toxin translocation peptide, e.g., to enablethe PA and/or mPA to recognize and transport the toxin into thenociceptor cell.

In some embodiments of all the aspects described herein, a translocationdomain can be a polycationic sequence. Such sequences are discussed inthe art; see, e.g., Blanke, Proc. Natl. Acad. Sci. USA 93, pp.8437-8442, 1996, and US Patent Application Publication No: US2003/0202989, each of which is incorporated here by reference in itsentirety. A polycationic sequence can comprise at least 2 cationic aminoacids, e.g., lysine, arginine, or histidine. In some embodiments of allthe aspects described herein that employ a polycationic sequence fortranslocation function, the polycationic sequence can comprise at leastabout 3, about 6, or about 8 cationic amino acids. In some embodimentsof all the aspects described herein that employ a polycationic sequencefor translocation function, the polycationic sequence can comprise thesequence KKK, KKKKKK (SEQ ID NO: 59), or KKKKKKKK (SEQ ID NO: 60). Insome embodiments of all the aspects described herein that employ apolycationic sequence for translocation function, the polycationicsequence can comprise the sequence HHH, HHHHHH (SEQ ID NO: 61), orHHHHHHHH (SEQ ID NO: 62). In some embodiments of all the aspectsdescribed herein that employ a polycationic sequence for translocationfunction, the polycationic sequence can comprise the sequence RRR,RRRRRR (SEQ ID NO: 63), or RRRRRRRR (SEQ ID NO: 64).

In some embodiments of toxin fusion proteins as described herein, afirst fusion protein domain comprises an anthrax toxin protectiveantigen (PA) moiety and a second fusion protein domain comprises ananthrax toxin translocation peptide fused with an inhibitor cysteineknot (ICK) toxin (e.g., a Conotoxin (CTx)). In other embodiments of afirst fusion protein domain, the the anthrax toxin translocation peptideis replaced with a clostridial neurotoxin translocation domain, H_(N),or a polycationic sequence.

In some embodiments of toxin fusion proteins as described herein, thefirst domain comprises a mutant anthrax toxin protective antigen (mPA)moiety that has been altered to block its native receptor-bindingfunction, fused with a molecule capable of specifically targeting anociceptor surface receptor or an ion channel receptor, and the seconddomain comprises an anthrax toxin translocation peptide fused with anintracellularly-acting toxin catalytic domain. In other embodiments of afirst fusion protein domain, the the anthrax toxin translocation peptideis replaced with a clostridial neurotoxin translocation domain, H_(N),or a polycationic sequence.

In some embodiments of toxin fusion proteins as described herein, afirst domain comprises: i) an anthrax toxin protective antigen (PA)moiety; or ii) a mutant anthrax toxin protective antigen (mPA) moietythat has been altered to block its native receptor-binding function,fused with a molecule capable of specifically targeting a nociceptorsurface receptor or an ion channel receptor; and a second domaincomprises an anthrax toxin edema factor (EF) and/or anthrax toxin lethalfactor (LF).

In another aspect, described herein is an engineered fusion proteincomprising an anthrax toxin Protective-Antigen (PA) moiety or itsreceptor binding domain (Pad4) fused with an inhibitor cysteine knot(ICK) toxin (e.g., a Conotoxin (CTx)).

In order to form a functional pore, PA and/or mPA must oligomerize.Accordingly, in some embodiments of any of the aspects described hereinthat involve a PA polypeptide, the PA or mPA can in an oligomeric form.In some embodiments of any of the aspects described herein that involvea PA polypeptide, the PA or mPA can be in an oligomeric form prior toadministration to a subject and/or prior to coming in contact with anociceptor cell.

Linkers

A linker may be used to connect two or more domains or portions of apolypeptide as described herein. Linker molecules (“linkers”) may bepeptides, which consist of one to multiple amino acids, or non-peptidemolecules. Examples of peptide linker molecules useful in thepolypeptides described herein include glycine-rich peptide linkers (see,e.g., U.S. Pat. No. 5,908,626), wherein more than half of the amino acidresidues are glycine. Preferably, such glycine-rich peptide linkersconsist of about 20 or fewer amino acids.

Linker molecules may also include non-peptide or partial peptidemolecules. For instance, the peptides can be linked to peptides or othermolecules using well known cross-linking molecules such asglutaraldehyde or EDC (Pierce, Rockford, Ill.).

In some embodiments of the fusion proteins described herein, the variousdomains and moieties, TM, TL, PAd4, PA fragments, LF, LFn, EF, EFn, mPA,various types of toxin (BTx including the various serotypes, TTx, ABtoxins, Ricin toxin, Cholera toxin, PE, Shiga toxin, DT, conotoxin, anagatoxin, a delta-palutoxin, a huwentotoxin or a ProTx II toxin) etc.are joined together in the respective fusion protein with a linkerpeptide. Examples of linker peptide include, but are not limited to:

(SEQ ID NO: 65) FHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTE; (SEQ ID NO: 66) VEIEDTE, (SEQ ID NO: 67) KDIRKILSGYIVEIEDTE;(SEQ ID NO: 68) STEGLLLNIDKDIRKILSGYIVEIEDTE, (SEQ ID NO: 69)EVKQENRLLNESES; and (SEQ ID NO: 70)VGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTE.

Flexible linkers are generally composed of small, non-polar or polarresidues such as Gly, Ser and Thr. In one embodiment of a fusion proteindescribed herein that includes a linker, the linker peptide comprises atleast one amino acid that is Gly or Ser. In one embodiment of a fusionprotein described herein that includes a linker, the linker is aflexible polypeptide between 1 and 25 residues in length. Commonexamples of flexible peptide linkers include (GGS)_(n), where n=1 to 8,or (Gly4Ser)n repeat where n=1-8 (SEQ ID NO:57), preferably, n=3, 4, 5,or 6, that is (Gly-Gly-Gly-Gly-Ser)n, where n indicates the number ofrepeats of the motif. For example, the flexible linker is (GGS)₂, GGSGGS(SEQ ID NO: 58).

In one embodiment of a fusion protein described herein that includes alinker, the linker peptide is 1-20 amino acids long. In one embodiment,the linker peptide is stable in human serum for at least 1 minute. Inone embodiment, the linker peptide does not comprise Lys and/or Arg.

In one embodiment of a fusion protein including a linker and PAd4, thelinker appended to the N-terminus of PAd4 is less than 20 amino acids inlength and is comprised of at least three amino acids Gly, Ser, and Ala.

In one embodiment of a fusion protein including a linker and PAd4, thelinker appended to the N-terminus of PAd4 is less than 20 amino acids inlength and is comprised of at least four amino acids Gly, Ser, Thr, andAla.

In one embodiment of a fusion protein including a linker, the linker isstable in human serum for at least 1 minute and is less than 20 aminoacids in length.

Bifunctional cross-linking molecules are linker molecules that possesstwo distinct reactive sites. For example, one of the reactive sites of abifunctional linker molecule may be reacted with a functional group on apeptide to form a covalent linkage and the other reactive site may bereacted with a functional group on another molecule to form a covalentlinkage. General methods for cross-linking molecules have been reviewed(see, e.g., Means and Feeney, Bioconjugate Chem., 1: 2-12 (1990)).

Homobifunctional cross-linker molecules have two reactive sites whichare chemically the same. Examples of homobifunctional cross-linkermolecules include, without limitation, glutaraldehyde;N,N′-bis(3-maleimido-propionyl-2-hydroxy-1,3-propanediol (asulfhydryl-specific homobifunctional cross-linker); certainN-succinimide esters (e.g., disuccinimidyl suberate,dithiobis(succinimidyl propionate), and soluble bis-sulfonic acid andsalt thereof (see, e.g., Pierce Chemicals, Rockford, Ill.; Sigma-AldrichCorp., St. Louis, Mo.).

Preferably, a bifunctional cross-linker molecule is a heterobifunctionallinker molecule, meaning that the linker has at least two differentreactive sites, each of which can be separately linked to a peptide orother molecule. Use of such heterobifunctional linkers permitschemically separate and stepwise addition (vectorial conjunction) ofeach of the reactive sites to a selected peptide sequence.Heterobifunctional linker molecules useful in the invention include,without limitation, m-maleimidobenzoyl-N-hydroxysuccinimide ester (see,Green et al., Cell, 28: 477-487 (1982); Palker et al., Proc. Natl. Acad.Sci (USA), 84: 2479-2483 (1987)); m-maleimido-benzoylsulfosuccinimideester; maleimidobutyric acid N-hydroxysuccinimide ester; andN-succinimidyl 3-(2-pyridyl-dithio)propionate (see, e.g., Carlos et al.,Biochem. J., 173: 723-737 (1978); Sigma-Aldrich Corp., St. Louis, Mo.).

PAd4 domains—In one embodiment of a fusion protein comprising a PAd4domain described herein, the fusion protein comprises about 2-10 PAd4domains in tandem, or 1-5 PAd4 domains, or 2-5 PAd4 domains, etc., intandem. In one embodiment of a fusion protein, approximately 1-60consecutive amino acids from the N-terminal side of PA adjacent to thenative PAd4 domain are further incorporated between the toxin moiety(e.g., BTx moiety, TTx moiety, disulfide-containing peptide toxinmoiety, AB toxin moiety, etc., and the PAd4 domain(s).

In one embodiment of a fusion protein comprising a PAd4 domain or acomposition comprising a PAd4 domain described herein, the fusionprotein further comprises domain 2 of PA (PAd2). The PAd2 domain can befound at amino acid residues 259-487 of the PA sequence (SEQ. ID. NO:1).In one embodiment of a fusion protein or a composition described herein,the fusion protein comprises, consists of, or consist essentially of thePAd2 and the PAd4 domain of PA. For example, the fusion proteincomprises, consists of, or consist essentially of the residues 259-487and 488-764 of PA (SEQ. ID. NO:1).

In one embodiment of a fusion protein comprising a PAd4 domain or acomposition comprising a PAd4 domain described herein, the fusionprotein further comprises a variant form of intact PA in which the furincleavage site has been ablated by mutation (M), so that the PA isresistant to proteolytic activation and hence does not oligomerize.

In one embodiment of a fusion protein comprising a PA, or a PA fragmentthereof, or a C-terminal receptor binding domain of PA that binds ANTXR2as described herein, or a composition comprising a fusion proteinincluding a PA or PA fragment thereof, or a C-terminal receptor bindingdomain of PA that binds ANTXR2 as described herein, the PA or PAfragment thereof, or a C-terminal receptor binding domain of PA thatbinds ANTXR2, the PA-derived protein is modified or mutated.

In one embodiment, the PAd4, the PA or PA fragment thereof, or aC-terminal receptor binding domain of PA that binds ANTXR2 is resistantto cleavage by a protease, such as Lys C. Other examples of proteasesthat the PA can be made resistant to include but are not limited tolysyl peptidase, trypsin, Enterokinase, clostripain, elastase,thermolysin, endoproteinase Lys-C, and endoproteinase Arg-C.

In one embodiment of any fusion protein described as including a PAd4domain, one or more of the Lys residues in the PAd4 domain at positions594, 613, 633, 637, 653, 673, 679, 680, 684, 695, 703, 722, 723, 729,and 730 has been replaced by Arg or His, wherein the numbering refers tothat of SEQ ID NO:1 after minusing the 29 aa signal peptide in SEQ. ID.NO:1. This includes any C-terminal receptor binding domain of a PA.

In one embodiment of any fusion protein described as including a PAd4domain, one or more of the Lys residues in the PAd4 domain at positions623, 642, 662, 666, 682, 702, 708, 709, 713, 724, 732, 751, 752, 758,and 759 in SEQ. ID. NO:1 has been replaced, for example, by Arg or His.

In one embodiment, one or more of the Asn residues in the PAd4 domain atposition 630, 742, and/or 748 of SEQ ID NO: 1 has been replaced by Asp.

PA In one embodiment of a fusion protein described herein comprising aPA domain or a composition comprising such a fusion protein, the PA is avariant or mutant form of PA that is resistant to furin cleavage(PA^(furin-)) or is mutated to block its native receptor-bindingfunction (mPA). In one embodiment, the PA furin cleavage site comprisingamino acid residues RKKR has been replaced by a furin-resistant aminoacid sequence. RKKR are the residues 164-167 of SEQ ID NO: 1 minus the29 amino acid signal peptide in SEQ ID NO:1. In one embodiment, thefurin-resistant amino acid sequence is SSSR (SEQ ID NO: 32) or SSSS (SEQID NO: 33). In one embodiment, PA has one or two mutations that blockthe receptor-binding function of PA, N711A and/or D712A; the amino acidnumbering is according to SEQ ID NO:1, the entire PA including the 29residue signal peptide. The two mutations are N682A/D683A in the PAsequence numbered without the 29 residue signal peptide.

In one embodiment of a fusion protein described herein comprising a PA,the PA is modified or mutated, for example, to be resistant to cleavageby a protease, such as Lys-C or furin. For example, the one or more ofthe Lys residues in the PAd4 domain of the PA at positions 594, 613,633, 637, 653, 673, 679, 680, 684, 695, 703, 722, 723, 729, and 730 canbe replaced by Arg or His. (SEQ ID NO: 1 after the removal of theN-terminal the 29 aa signal peptide) (This sequence is P13423 with the29 aa signal peptide). In other words, one or more, up to and includingeach of the Lys residues in the PAd4 domain of PA, at positions 623,642, 662, 666, 682, 702, 708, 709, 713, 724, 732, 751, 752, 758, and 759in SEQ. ID. NO:1 can be replaced, for example, by Arg or His.

In one embodiment of a fusion protein described herein, the fusionprotein further comprises at least one D-amino acid at the N-terminus ofthe fusion protein.

In one embodiment of a fusion protein described herein, the fusionprotein is glycosylated.

In one embodiment of a fusion protein described herein, the fusionprotein is non-glycosylated.

Production of Engineered Fusion Protein

The various polypeptides (e.g. fusion polypeptides or first or secondpolypeptides) described herein can be purified from natural sourcesand/or produced recombinantly using any method that is known in the art.For examples, the engineered fusion proteins described herein can beproduced by recombinant molecular cloning that is known in the art. Byway of non-limiting example, PA can be purified from the Sterne strainof B. anthracis or synthesized by other known means. In B. anthracis,the gene for PA is located on a plasmid referred to as pXO1 (Milne etal., 1994, J. of Biol. Chem. 269(32):20607-20612; which is incorporatedby reference herein in its entirety). Methods of recombinant expressionare well-known in the art. In some embodiments, PA63 can be substitutedfor full-length PA. The PA63 fragment may be purified fromtrypsin-treated PA by anion exchange chromatography (Milne et al., 1994,supra). PA encoding gene has been cloned and sequenced (Vodkin, et al.,1983, Cell 34:693-697; which is incorporated by reference herein in itsentirety) and may be used to obtain purified PA polypeptide.

The fusion of two polypeptide moieties is effected either by recombinantDNA technology, or using a sortase reaction (see e.g., WO 2012096926,WO2013177231, WO2014088928,; U.S. Pat. No. 9,079,952, and US PatentApplication Publications No: US 2013/0336974 and US 2015/0267186, eachof which is incorporated by reference herein in its entirety.), or othermethod of chemical linking/biochemical conjugation that is known in theart.

Recombinant DNA and molecular biology techniques can be use to producethe described engineered fusion protein. The process of cloning the cDNAsegments and sequences that encode the respective protein domains andmoieties, e.g., TM, TL, PAd4, PA fragments, LF, LFn, EF, EFn, mPA,various types of toxin (BTx, TTx, AB toxins, Ricin toxin, Cholera toxin,PE, Shiga toxin, DT, conotoxin, an agatoxin, a delta-palutoxin, ahuwentotoxin or a ProTx II toxin) etc., the production of DNA sequencesencoding the various peptide linkers, the ligation of different cDNAsequences, the construction of the expression vectors (e.g., plasmid,bacteriophage, phagmid, or viral vector) for the various engineeredfusion proteins, and the protein expression and purification of variousrecombinant engineered fusion proteins can be performed by conventionalrecombinant molecular biology and protein biochemistry techniques suchas described in Lewin's Genes XI, published by Jones & BartlettPublishers, 2014 (ISBN-1449659055); Michael Richard Green and JosephSambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN1936113414); Davis et al., Basic Methods in Molecular Biology, ElsevierScience Publishing, Inc., New York, USA (2012) (ISBN 044460149X);Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel(ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385),Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), JohnWiley and Sons, Inc., 2005, which are all incorporated by referenceherein in their entireties.

The engineered fusion proteins and peptide linkers can be produced byany method known in the art for the synthesis of a fusion protein, inparticular, by chemical synthesis or by recombinant expressiontechniques.

The engineered fusion proteins of the invention can be produced by anymethod known in the art for the expression and purification ofrecombinant proteins.

Recombinant expression of engineered fusion proteins requiresconstruction of an expression vector containing a polynucleotide thatencodes the engineered fusion protein described herein. Thepolynucleotide can further sequences that encode additional amino acidsfor the purpose of protein purification or identifying or locating theengineered fusion protein in the expression system or during the proteinpurification process. Once a polynucleotide encoding an engineeredfusion protein has been obtained, the vector for the production of thefusion protein can be produced by recombinant DNA technology usingtechniques well known in the art. Thus, methods for preparing a proteinby expressing a polynucleotide containing a fusion protein-encodingnucleotide sequence are described herein. Methods which are well knownto those skilled in the art can be used to construct expression vectorscontaining protein coding sequences and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. The invention, thus, provides replicable vectorscomprising a nucleotide sequence encoding a fusion protein of theinvention, operably linked to a promoter. The expression vector istransferred to a host cell by conventional techniques and thetransfected cells are then cultured by conventional techniques toproduce an engineered fusion protein of the invention. Thus, theinvention includes host cells containing a polynucleotide encoding afusion protein, operably linked to a heterologous promoter.

A variety of host-expression vector systems can be utilized to expressthe fusion proteins of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest can beproduced and subsequently purified, but also represent cells which can,when transformed or transfected with the appropriate nucleotide codingsequences, express the fusion protein of the invention in situ. Theseinclude but are not limited to microorganisms such as prokaryoticbacteria (e.g., attenuated Bacillus anthracis strains, E. coli, B.subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA orcosmid DNA expression vectors containing the fusion protein codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing the fusion proteincoding sequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the fusion proteincoding sequences; plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or transformed with recombinant plasmid expression vectors(e.g., Ti plasmid) containing the fusion protein coding sequences; ormammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter). Bacterial cells such as E. coli and attenuated B.anthracis strains are especially useful for the expression of the fusionproteins described herein.

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the fusionprotein being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of a fusion protein, vectors which direct the expression ofhigh levels of a fusion protein products that are readily purified canbe desirable. Such vectors include, but are not limited, to the E. coliexpression vector pUR278 (Ruther et al., EMBO J., 2:1791 (1983)), inwhich the fusion protein coding sequence can be ligated individuallyinto the vector in frame with the lacZ coding region so that a fusionprotein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.,13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem., 24:5503-5509(1989)); and the like pGEX vectors can also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption and binding to matrix glutathione-agarosebeads followed by elution in the presence of free glutathione. The pGEXvectors are designed to include thrombin or factor Xa protease cleavagesites so that the cloned target gene product can be released from theGST moiety. Alternately, the pET expression vectors can be used forproducing histidine-tagged recombinant proteins, where thehistidine-tagged recombinant proteins can be affinity purified by anickel column. Expression of recombinant proteins in Pichia pastoris isdescribed by Holliger, P. (2002) Meth. Mol. Biol., 178:349-57, and ishereby incorporated by reference. Examples of recombinant expression andpurification of engineered anthrax containing fusion proteins and BTx-or TTx-containing fusion proteins are known in the art, and aredescribed in more detail in, e.g., International Patent Publication WO2012/096926 and WO 2015/166242; U.S. Pat. Nos. 9,079,952, 9,234,011,9,243,301, and US Patent Application Publications No: US 2013/0336974,US 2015/0267186, US 2015/0044210, each of which is incorporated byreference herein in its entirety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The fusion protein coding sequence can becloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

Large scale expression of heterologous proteins in the algaeChlamydomonas reinhardtii are described by Griesbeck C. et. al. 2006Mol. Biotechnol. 34:213-33; Manuell A L et. al. 2007 Plant Biotechnol J.Eprint; Franklin S E and Mayfield S P, 2005, Expert Opin Biol Ther. Feb;5(2):225-35; Mayfield S P and Franklin S E, 2005 Vaccine March 7;23(15):1828-32; and Fuhrmann M. 2004, Methods Mol Med. 94:191-5. Foreignheterologous coding sequences are inserted into the genome of thenucleus, chloroplast and mitochondria by homologous recombination. Thechloroplast expression vector p64 carrying the most versatilechloroplast selectable marker aminoglycoside adenyl transferase (aadA),which confers resistance to spectinomycin or streptomycin, can be usedto express foreign protein in the chloroplast. Biolistic gene gun methodis used to introduce the vector in the algae. Upon its entry intochloroplasts, the foreign DNA is released from the gene gun particlesand integrates into the chloroplast genome through homologousrecombination.

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the coding sequence of fusion protein can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the fusion protein in infected hosts. See, e.g., Logan &Shenk, Proc. Natl. Acad. Sci. USA, 81:355-359 (1984). Specificinitiation signals can also be required for efficient translation ofinserted fusion protein coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression canbe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see, Bittner et al., Methodsin Enzymol., 153:51-544 (1987)).

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,NSO, 293, 3T3, W138, and in particular, breast cancer cell lines suchas, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammarygland cell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe fusion protein can be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells can beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method canadvantageously be used to engineer cell lines which express the fusionprotein. Such engineered cell lines can be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the fusion protein.

A number of selection systems can be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell, 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA, 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell, 22:817 (1980)) genes canbe employed in tk-, hgprt- or aprt-cells, respectively. Also,anti-metabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Proc. Natl. Acad. Sci. USA, 77:357 (1980); O'Hare et al., Proc.Natl. Acad. Sci. USA, 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418; Wuand Wu, Biotherapy, 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.Toxicol., 32:573-596 (1993); Mulligan, Science, 260:926-932 (1993); andMorgan and Anderson, Ann. Rev. Biochem., 62:191-217 (1993); Can, 1993,TIB TECH 11(5):155-215); and hygro, which confers resistance tohygromycin (Santerre et al., Gene, 30:147 (1984)). Methods commonlyknown in the art of recombinant DNA technology can be routinely appliedto select the desired recombinant clone, and such methods are described,for example, in Current Protocols in Molecular Biology, Ausubel et al.,eds. (John Wiley & Sons, NY 1993); Kriegler, Gene Transfer andExpression, A Laboratory Manual (Stockton Press, NY 1990); and CurrentProtocols in Human Genetics, Dracopoli et al., eds. (John Wiley & Sons,NY 1994), Chapters 12 and 13; Colberre-Garapin et al., J. Mol. Biol.,150:1 (1981), which are incorporated by reference herein in theirentireties.

The expression levels of an engineered fusion protein described hereincan be increased by vector amplification (for a review, see Bebbingtonand Hentschel, The use of vectors based on gene amplification for theexpression of cloned genes in mammalian cells in DNA cloning, Vol. 3.(Academic Press, New York, 1987)). When a marker in the vector systemexpressing the fusion protein is amplifiable, increase in the level ofinhibitor present in culture of host cell will increase the number ofcopies of the marker gene. Since the amplified region is associated withthe nucleic acid sequence encoding the engineered fusion proteindescribed herein, production of the engineered fusion protein will alsoincrease (Crouse et al., Mol. Cell. Biol., 3:257 (1983)).

The host cell can be co-transfected with two expression vectors of theinvention, the first vector encoding a first fusion protein and thesecond vector encoding a second fusion protein. The two vectors cancontain identical selectable markers which enable equal expression of afusion polypeptides. Alternatively, a single vector can be used whichencodes, and is capable of expressing, both fusion polypeptides. Abi-cistronic expression cassette encoding both fusion polypeptides isinserted into the expression vector (Proudfoot, Nature, 322:52 (1986);Kohler, Proc. Natl. Acad. Sci. USA, 77:2197 (1980)).

Once a fusion protein of the invention has been produced by an animal orrecombinantly expressed, it can be purified by any method known in theart for protein purification for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the engineered fusion proteindescribed herein can be fused to heterologous polypeptide sequencesdescribed herein or otherwise known in the art, to facilitatepurification.

For the purpose of affinity purification, relevant matrices for affinitychromatography, such as glutathione-, amylase-, and nickel- orcobalt-conjugated resins are used. Many of such matrices are availablein “kit” form, such as the Pharmacia GST purification system and theQIAexpress™ system (QIAGEN®) useful with histidine-tagged fusionproteins. Tags can also facilitate the detection of the expressedrecombinant fusion proteins. Examples of such tags include the variousfluorescent proteins (e.g., GFP) as well as “epitope tags,” which areusually short peptide sequences for which a specific antibody isavailable. Well known epitope tags for which specific monoclonalantibodies are readily available include FLAG, influenza virushaemagglutinin (HA), and c-myc tags. In some cases, the fusion domainshave a protease cleavage site, such as for Factor Xa or Thrombin, whichallows the relevant protease to partially digest the fusion proteins andthereby liberate the recombinant proteins therefrom. The liberatedproteins can then be isolated from the fusion domain by subsequentchromatographic separation.

Accordingly, in one embodiment, encompassed herein is a nucleic acidsequence encoding any one of the fusion proteins described herein.

In one embodiment, provided herein is a vector comprising a nucleic acidsequence encoding any one of the fusion protein described herein. Forexamples, the vector can be a plasmid, a bacteriophage, a phagmid, acosmid, a viral vector, or a viral particle. These vectors are known inthe art. In one embodiment, provided herein is a plasmid comprising anucleic acid sequence encoding any one of the fusion protein describedherein. For example, the plasmid is a bacterial plasmid. In oneembodiment of a vector described, the vector is an expression vector.For example, the plasmid (vector) is an expression plasmid for therecombinant protein expression in a bacteria, e.g., Escherichia coli. Inone embodiment of an expression vector described, the expression vectoris a bacterial expression vector. In one embodiment of an expressionvector described, the expression vector is a prokaryotic expressionvector. In one embodiment of an expression vector described, theexpression vector is an eukaryotic expression vector. In one embodimentof an expression vector described, the expression vector is a mammalianexpression vector. In one embodiment, the expression vector is a yeastexpression vector.

In another embodiment, provided herein is a viral particle comprising avector comprising a nucleic acid described in the preceding paragraph.In another aspect, provided herein is a viral particle comprising a anucleic acid described in the preceding paragraph.

In one embodiment, provided herein is a cell comprising a nucleic acidsequence encoding any one of the fusion proteins described herein or avector comprising a nucleic acid sequence encoding any one of the fusionproteins described herein. The cell can be a bacteria, a yeast cell, amammalian cell etc. For example, an E. coli carrying a plasmid thatcomprises a nucleic acid encoding a fusion protein described herein. Forexample, for the recombinant protein expression of the a fusion proteinencoded in the nucleic acid.

In another aspect, provided herein is a cell comprising a viral particlecomprising a vector comprising a nucleic acid sequence encoding any oneof the fusion proteins described herein. In another aspect, providedherein is a cell comprising a plasmid comprising a nucleic acid sequenceencoding any one of the fusion proteins described herein. In anotheraspect, provided herein is a cell comprising a viral particle comprisinga nucleic acid sequence encoding any one of the fusion proteinsdescribed herein.

In one embodiment, provided herein is a method of producing any one ofthe fusion proteins described herein, comprising (a) culturing a cellcomprising a nucleic acid sequence encoding any one of the fusionproteins described herein, or a vector (e.g., a plasmid) comprising anucleic acid sequence encoding any one of the fusion proteins describedherein, or a viral particle comprising a vector comprising a nucleicacid sequence encoding any one of the fusion proteins described, or aviral particle comprising a nucleic acid sequence encoding any one ofthe fusion proteins described herein herein, wherein the culturing isperformed under conditions such that the fusion protein described isexpressed; and (b) recovering the fusion protein.

In one embodiment, provided herein is a fusion protein produced by themethod described herein, specifically, the method comprising (a)culturing a cell comprising a nucleic acid sequence encoding any one ofthe fusion proteins described herein or a vector (e.g., a plasmid)comprising a nucleic acid sequence encoding any one of the fusionproteins described herein, or a viral particle comprising a vectorcomprising a nucleic acid sequence encoding any one of the fusionproteins described, or a viral particle comprising a nucleic acidsequence encoding any one of the fusion proteins described hereinherein, wherein the culturing is performed under conditions such thatthe fusion protein described is expressed; and (b) recovering the fusionprotein.

In one embodiment of the method described for producing a fusionprotein, the cell is a prokaryotic cell such as bacteria. In oneembodiment of the method described for producing a fusion protein, thecell is a bacteria cell. In one embodiment, the bacteria is Escherichiacoli (E. coli). In another embodiment, the bacteria is an attenuated B.anthracis strains (e.g. CDC 684).

In one embodiment of a method of producing a fusion protein describedherein, the cell is a yeast cell. In one embodiment, the yeast isSaccharomyces cerevisiae. In one embodiment, the yeast is cellglycosylation deficient.

In one embodiment of a method of producing a fusion protein describedherein, the yeast is glycosylation and protease deficient. In oneembodiment, the protease is furin or furin-like protease.

In one embodiment of a method of producing a fusion protein describedherein, the cell is a mammalian cell. In one embodiment, the mammaliancell is a COS cell, a CHO cell, or an NSO cell.

Compositions

In one aspect, described herein is a composition comprising at least oneengineered fusion protein as described herein. In one embodiment, thecomposition further comprises a pharmaceutically acceptable carrier,excipient or diluent.

In some aspects, any compositions described in the preceding paragraphsor any compositions comprising a fusion protein described in thepreceding paragraphs is use for the treatment of pain. Treatment of paincan include administering more than one, i.e., several, of the differentcompositions described in the preceding paragraphs. For example,compositions comprising fusion proteins comprising LFn, LF, EFn, or EF,and where there is nociceptor receptor binding protein present in thefusion protein, the composition would preferably be used in combinationwith a composition comprising a PA, a PA fragment, a PAd4 containingfragment of PA, or a C-terminal receptor binding domain of PA of PA.

In some embodiments, the composition can comprise two or more differentengineered fusion proteins, e.g., fusion proteins with different firstand/or second domains. Such mixtures of proteins can, e.g. reduceoff-target effects and/or provide multiple mechanisms of inhibitingnociceptors to increase efficacy. In some embodiments, the two or moredifferent engineered fusion proteins can be in oligomeric form such thatthe oligomeric complex comprises at least two different engineeredfusion proteins. In some embodiments, a first engineered fusion proteinhas a first domain comprising an anthrax toxin protective antigen (PA)moiety; and a second engineered fusion protein has a first domaincomprising a mutant anthrax toxin protective antigen (mPA) moiety thathas been altered to block its native receptor-binding function fusedwith a molecule capable of specifically targeting a nociceptor surfacereceptor or an ion channel receptor. In some embodiments, a compositionas described herein can further comprise a pharmaceutically acceptablecarrier or excipient.

When a polypeptide comprises an anthrax toxin translocation peptide, thetranslocation peptide can cause the polypeptide to be bound by andtranslocated across a membrane by PA and/or mPA present. Accordingly,the PA and/or mPA and the polypeptide to be translocated (e.g. a toxin)can be present as separate polypeptides. In one aspect then, describedherein is a composition comprising:

(I) a first polypeptide selected from the group consisting of:

-   -   a) an anthrax toxin protective antigen (PA) moiety; and        optionally    -   b) a mutant anthrax toxin protective antigen (mPA) moiety that        has been altered to block its native receptor-binding function,        fused with a molecule capable of specifically targeting a        nociceptor surface receptor or an ion channel receptor; and

(II) a second polypeptide selected from the group consisting of:

-   -   c) an anthrax translocation signal peptide fused with an        inhibitor cysteine knot (ICK) toxin (e.g., a Conotoxin (CTx));    -   d) an anthrax translocation signal peptide fused with an        intracellularly-acting toxin catalytic domain; and/or    -   e) an anthrax toxin edema factor (EF) and/or anthrax toxin        lethal factor (LF).

In one embodiment of such a composition, the first polypeptide comprisesan anthrax toxin protective antigen (PA) moiety and the secondpolypeptide comprises an anthrax toxin translocation peptide fused withan inhibitor cysteine knot (ICK) toxin (e.g., a Conotoxin (CTx)).

In another embodiment of such a composition, the first polypeptidecomprises a mutant anthrax toxin protective antigen (mPA) moiety thathas been altered to block its native receptor-binding function, fusedwith a molecule capable of specifically targeting a nociceptor surfacereceptor or an ion channel receptor, and the second polypeptidecomprises an anthrax toxin translocation peptide fused with anintracellularly-acting toxin catalytic domain. In other embodiments of asecond polypeptide, the the anthrax toxin translocation peptide isreplaced with a clostridial neurotoxin translocation domain, H_(N), or apolycationic sequence.

In another embodiment of such a composition, the first polypeptidecomprises: i) an anthrax toxin protective antigen (PA) moiety; or ii) amutant anthrax toxin protective antigen (mPA) moiety that has beenaltered to block its native receptor-binding function fused with amolecule capable of specifically targeting a nociceptor surface receptoror an ion channel receptor; and the second polypeptide comprises ananthrax toxin edema factor (EF) and/or anthrax toxin lethal factor (LF).

In another embodiment of such a composition comprising a first andsecond polypeptide, the PA or mPA is in an oligomeric form. In anotherembodiment of such a composition comprising first and secondpolypeptides, the composition comprises both i) an anthrax toxinprotective antigen (PA) moiety and ii) a mutant anthrax toxin protectiveantigen (mPA) moiety that has been altered to block its nativereceptor-binding function, fused with a molecule capable of specificallytargeting a nociceptor surface receptor or an ion channel receptor. Inone embodiment, the i) anthrax toxin protective antigen (PA) moiety andii) mutant anthrax toxin protective antigen (mPA) moiety that has beenaltered to block its native receptor-binding function fused, with amolecule capable of specifically targeting a nociceptor surface receptoror an ion channel receptor, two different first polypeptides are inoligomeric form such that the oligomeric complex comprises bothpolypeptides. In one embodiment, the composition further comprises apharmaceutically acceptable carrier or excipient.

In one embodiment of any of the aspects described herein, thecomposition further comprises a native anthrax toxin protective antigen(PA) protein. In one embodiment, the PA protein is an oligomeric PA. Inanother embodiment, the oligomeric PA is bound to the fusion protein.

In one embodiments of any of the compositions comprising LFn, LF, EF orEFn-containing fusion proteins described herein, the composition furthercomprises a native anthrax toxin protective antigen (PA) protein. In oneembodiment, the PA protein is an oligomeric PA. In another embodiment,the oligomeric PA is bound to the fusion protein.

Uses of Engineered Fusion Proteins and Compositions for Pain Treatment

Any compositions comprising a fusion protein described herein, usedindividually or in combinations, can be used for the treatment of pain.Similarly, any fusion protein described herein can be used for thetreatment of pain. Moreover, it is envisioned that various combinationsof the described compositions or various combinations of the describedengineered fusion proteins would be used in the treatment of pain. Thefusion proteins and compositions described herein can selectively bindto nociceptors and deliver toxins that kill and/or inhibit thenociceptor cells. In some embodiments, other neurons are not affected bythe fusion proteins and/or compositions described herein.

In another aspect, provided herein is a method of manufacture of apharmaceutical composition comprising one or more of the fusion proteinsdescribed in the preceding paragraphs and a pharmaceutically acceptablecarrier or excipient.

In another aspect, provided herein is a fusion protein described in thepreceding paragraphs for use in the manufacture of medicament for thetreatment of pain. In one embodiment, the fusion protein is formulatedwith at least one pharmaceutically acceptable carrier or excipient.

In another aspect, provided herein is a fusion protein described in thepreceding paragraphs for use in the treatment of pain. In oneembodiment, the fusion protein is formulated with at least onepharmaceutically acceptable carrier or excipient.

Accordingly, in one aspect, described herein is a method for treatmentof pain, the method comprising administering to a subject in needthereof an effective, pain reducing amount of a composition as describedherein. More than one composition can be administered, eithersimultaneously or sequentially. For example, compositions comprisingfusion proteins comprising LFn, LF, EFn, or EF, and where there isnociceptor receptor binding protein present in the fusion protein, thecomposition would preferably be used in combination with a compositioncomprising a PA, a PA fragment, a PAd4 containing fragment of PA, or aC-terminal receptor binding domain of PA of PA.

In another aspect, described herein is a method for treatment of pain,the method comprising administering to a subject in need thereof aneffective, pain reducing amount of a fusion protein comprising: (a) abotulinum neurotoxin (BTx) or a tetanus neurotoxin (TTx), and (b) ananthrax toxin protective antigen (PA), or a C-terminal receptor-bindingdomain of PA, wherein part (a) and (b) are linked or fused together, ora composition comprising the fusion protein.

In another aspect, described herein is a method for treatment of pain,the method comprising administering to a subject in need thereof aneffective, pain reducing amount of a fusion protein comprising: (a) anon-cytotoxic protease, which protease is capable of cleaving a SNAREprotein in a nociceptor neuron; and (b) a protein capable of binding toan anthrax toxin protective antigen (PA) or a fragment thereof, whereinthe PA or PA fragment thereof binds a receptor expressed on thenociceptor neuron, or a composition comprising the fusion protein.

In another aspect, described herein is a method for treatment of pain,the method comprising administering to a subject in need thereof aneffective, pain reducing amount of a fusion protein comprising (a) adisulfide-containing peptide toxin which is capable of blocking ionchannels in a nociceptor neuron; and (b) a targeting moiety (TM) that iscapable of binding to a binding site on the nociceptor neuron, whereinthe nociceptor neuron expresses the ion channels therein, or acomposition comprising the fusion protein.

In another aspect, described herein is a method for treatment of pain,the method comprising administering to a subject in need thereof aneffective, pain reducing amount of a fusion protein comprising: (a) adisulfide-containing peptide toxin which is capable of blocking sodiumor calcium or both sodium and calcium channels in a nociceptor neuron;and (b) a protein capable of binding to an anthrax toxin protectiveantigen (PA) or a PA fragment that binds a receptor expressed on thenociceptor neuron, or a composition comprising the fusion protein.

In another aspect, described herein is a method for treatment of pain,the method comprising administering to a subject in need thereof aneffective, pain reducing amount of a fusion protein comprising: (a) anAB toxin; (b) an anthrax toxin protective antigen (PA) or a fragmentthereof, wherein the PA or fragment thereof binds a receptor expressedon a nociceptor neuron; and (c) a translocation domain (TL) that iscapable of translocating the toxin (a protease) from within an endosome,across the endosomal membrane and into the cytosol of the nociceptorneuron, or a composition comprising the fusion protein.

In another aspect, described herein is a method for treatment of pain,the method comprising administering to a subject in need thereof aneffective, pain reducing amount of a fusion protein comprising abotulinum neurotoxin (BTx) moiety comprising an N-terminal enzymaticdomain (LC or L chain) and an intermediatepore-forming/translocation-domain (H_(N)) of the BTx, linked to aC-terminal receptor-binding domain of anthrax toxin protective antigen(PA), or a composition comprising the fusion protein.

In another aspect, described herein is a method for treatment of pain,the method comprising administering to a subject in need thereof aneffective, pain reducing amount of a fusion protein comprising: (a) abotulinum neurotoxin N-terminal enzymatic domain of a botulinumneurotoxin (BTx) moiety, and (b) an N-terminal domain of anthrax toxinlethal factor (LFn), which domain binds to oligomeric forms of PA63, theproteolytically activated form of anthrax PA; or the N-terminal domainof anthrax toxin edema factor (EFn), which domain binds to oligomericforms of PA63, the proteolytically activated form of anthrax PA, whereinpart (a) is linked N-terminally or C-terminally or both N-terminally andC-terminally to part (b), or a composition comprising the fusionprotein.

In another aspect, described herein is a method for treatment of pain,the method comprising administering to a subject in need thereof aneffective, pain reducing amount of a fusion protein comprising anthraxtoxin protective antigen (PA), an anthrax toxin protective antigenC-terminal receptor binding domain (PAd4), or a nociceptorneuron-binding protein, linked to a disulfide-containing peptide toxin,or a composition comprising the fusion protein.

In another aspect, described herein is a method for treatment of pain,the method comprising administering to a subject in need thereof aneffective, pain reducing amount of a fusion protein comprising adisulfide-containing peptide toxin operably linked N-terminally orC-terminally or both N-terminally and C-terminally, or chemicallycrosslinked at one or more sites, to the N-terminal domain (LFn) ofanthrax toxin lethal factor, which domain binds to oligomeric forms ofPA63, the proteolytically activated form of anthrax PA; or theN-terminal domain (EFn) of anthrax toxin edema factor, which domainbinds to oligomeric forms of PA63, or a composition comprising thefusion protein.

In another aspect, described herein is a method for treatment of pain,the method comprising administering to a subject in need thereof aneffective, pain reducing amount of a fusion protein comprising an ABtoxin fused to a linker peptide linked to a C-terminal receptor-bindingdomain of anthrax toxin protective antigen (PAd4 domain), wherein thefusion protein further comprises a translocation domain, a holotoxin, ora mutant form of the holotoxin that have been modified (e.g.,chemically) or mutated to negate the toxin receptor-binding function ofthe AB toxin, or a composition comprising the fusion protein.

In another aspect, described herein is a method for treatment of pain,the method comprising administering to a subject in need thereof aneffective, pain reducing amount of a fusion protein comprising anN-terminal enzymatic domain (Chain A) together with atranslocation/pore-forming domain from a Clostridial neurotoxin or anon-Clostridial botulinum-like toxin, linked to a C-terminalreceptor-binding domain of anthrax toxin protective antigen (PAd4domain), or a composition comprising the fusion protein.

In another aspect, described herein is a method for treatment of pain,the method comprising administering to a subject in need thereof aneffective, pain reducing amount of an engineered fusion proteincomprising a native protective antigen (PA) or a mutant PA (mPA),wherein the mPA has been modified (e.g., chemically) or mutated so as toblock its native receptor-binding function, and a molecule that cantarget nociceptor neuron surface molecules, specifically in combinationwith anthrax toxin edema factor (EF) and/or anthrax lethal factor (LF),or a composition comprising the engineered fusion protein.

In another aspect, described herein is a method for treatment of pain,the method comprising administering to a subject in need thereof aneffective, pain reducing amount of an engineered fusion proteincomprising an anthrax protective antigen (PA) moiety fused with amolecule capable of specifically targeting a nociceptor surface receptoror a nociceptor ion channel receptor, and an anthrax lethal factordomain (LFn) fused to an intracellularly acting toxin catalytic domain.ANTXR2 is the native receptor for PA, or a composition comprising theengineered fusion protein.

In another aspect, described herein is a method for treatment of pain,the method comprising administering to a subject in need thereof aneffective, pain reducing amount of an engineered fusion proteincomprising a mutant anthrax protective antigen (mPA) moiety that hasbeen altered to block its native receptor-binding function, fused with amolecule capable of specifically targeting a nociceptor surface receptoror a nociceptor ion channel receptor, and an anthrax lethal factordomain (LFn) fused to an intracellularly acting toxin catalytic domain.ANTXR2 is the native receptor for PA, or a composition comprising theengineered fusion protein.

In another aspect, described herein is a method for treatment of pain,the method comprising administering to a subject in need thereof aneffective, pain reducing amount of an engineered fusion proteincomprising an anthrax toxin Protective-Antigen (PA) moiety or itsreceptor binding domain (PAd4) fused with an inhibitor cysteine knot(ICK) toxin, e.g., a Conotoxin (CTx), or a composition comprising theengineered fusion protein.

In another aspect, described herein is a method for treatment of pain,the method comprising administering to a subject in need thereof aneffective, pain reducing amount of an engineered fusion proteincomprising an anthrax toxin lethal factor domain (LFn) fused with aninhibitor cysteine knot (ICK) toxin and a Protective-Antigen (PA)moiety, or a composition comprising the engineered fusion protein.

In another aspect, described herein is a method for treatment of pain,the method comprising administering to a subject in need thereof aneffective, pain reducing amount of an engineered fusion proteincomprising an anthrax toxin lethal factor domain (LFn) fused with a Lchain of a Clostridial neurotoxin and a Protective-Antigen (PA) moiety,or a composition comprising the engineered fusion protein. In oneembodiment, this fusion protein can further comprise the belt of the Hchain of the Clostridial neurotoxin, the belt is the N-terminal segmentof the H chain.

Non-limiting examples of pain that can be treated according to themethods described herein can include: chronic pain; chronic neuropathicpain; painful diabetic neuropathy (PDN), post-herpetic neuropathy (PHN);trigeminal neuralgia (TN); inflammatory pain; neuropathic pain; achannelopathy; primary erythermalgia (PE); paroxysmal extreme paindisorder (PEPD); spinal cord injury pain; multiple sclerosis pain;phantom limb pain; post-stroke pain; chronic back pain; osteoarthritispain; cancer-associated pain; HIV-associated pain; chronic inflammatorypain; central neuropathy; peripheral neuropathy; anaesthesia dolorosa;hyperalgesia; hyperpathia; paresthesia; psychogenic pain; back pain;breakthrough pain; erythromelalgia; nerve compression and/or entrapment[e.g., carpal tunnel syndrome, tarsal tunnel syndrome, ulnar nerveentrapment, compression radiculopathy, radicular low back pain, spinalroot lesions, spinal root compression, lumbar spinal stenosis, sciaticnerve compression, and/or intercostal neuralgia]; neuritis; pain fromchemotherapy; chronic alcoholism (alcoholic polyneuropathy); rheumatoidarthritis pain; pain associated with burns; encephalitis pain; bonefracture pain; neuritis pain; autoimmune disease pain; postoperativepain; dental pain; pain associated with bacterial infection, e.g. abacterial infection or viral infection; pain associated withradiotherapy; pain associated with gout and irritable bowel syndrome;pain from trauma (such as from lacerations, incisions, burns, foreignbodies or bullet and/or shrapnel injuries, spinal cord injury, brachialplexus avulsion, nerve crush and/or entrapment; nerve transection;visceral pain (such as renal or ureteral colic, irritable bowelsyndrome, angina or cardiac pain, cardiac arrhythmia, period pain,interstitial cystitis, rectal pain, pain associated with diarrhoea,appendicitis, cholecystitis and pancreatitis); uremia pain; painassociated with hypothyroidism; pain associated with vitamin deficiency;headache pain (e.g., tension headache, migraine and cluster headache);idiopathic pain (e.g., trigeminal neuralgia, a complex regional painsyndrome [e.g. complex regional pain syndrome I and/or complex regionalpain syndrome II], allodynia or fibromyalgia); respiratory pain (e.g.,pain associated with asthma, airway hyper-reactivity in asthma, chroniccough, e.g. in asthma and/or chronic obstructive pulmonary disorder);fibromyalgia; hormonal therapy pain; hypothyroidism pain; epilepticpain; ataxia; periodic paralysis; acute itch and/or chronic itch pain.

In one embodiment, the pain to treat by using any of the compositionsdescribed herein is selected from diabetic neuropathic pain, cancerpain, fibromyalgia and other systemic pain disorders.

In one embodiment, the pain to treat by using any of the compositionsdescribed herein is selected from nerve, joint, skin, visceral, bladder,and muscle pain.

In one embodiment, the composition to be administered comprises a firstpolypeptide (or fusion protein) and a second polypeptide (or fusionprotein), and the first polypeptide is bound to the second polypeptidebefore administration.

In another aspect, described herein is a method for treatment of pain,the method comprising administering to a subject in need thereof aneffective, pain reducing amount of a first composition comprising

-   -   a) an anthrax toxin protective antigen (PA) moiety; and/or    -   b) a mutant anthrax toxin protective antigen (mPA) moiety that        has been altered to block its native receptor-binding function        fused with a molecule capable of specifically targeting a        nociceptor surface receptor or an ion channel receptor; and    -   c) a second composition comprising:        -   (i) an anthrax toxin translocation signal peptide fused with            an inhibitor cysteine knot (ICK) toxin (e.g., a Conotoxin            (CTx));        -   (ii) an anthrax toxin translocation peptide fused with an            intracellularly-acting toxin catalytic domain; and/or        -   (iii) an anthrax toxin edema factor (EF) and/or anthrax            toxin lethal factor (LF).

In any of the aspects drawn to a method of treating pain, theadministering can be performed by intrathecal infusion,intra-cerebroventricular infusion, an epidural injection into thecentral nervous system, or by peripheral administration usingintradermal injection, subcutaneous injection, intramuscular injection,intraneural injection, or intra-articular injection.

Accordingly, in one embodiment, described herein is a method fortreatment of nerve, joint, skin, visceral, bladder, or muscle paincomprising administering peripherally by intradermal injection,subcutaneous injection, intramuscular injection, intraneural injection,or intra-articular injection to a subject in need thereof an effective,pain reducing amount of a composition as described herein.

In another embodiment, described herein is a method for treatment ofdiabetic neuropathic pain, cancer pain, fibromyalgia or other systemicpain disorders comprising administering by epidural injection,intrathecal infusion or intra-cerebroventricular infusion into thecentral nervous system of a subject in need thereof an effective, painreducing amount of a composition as described herein.

In one embodiment of any of the methods of treating pain describedherein, the effective, pain reducing amount of a composition asdescribed herein is administered separately before, simultaneously, orafter administering a composition comprising an anthrax protectiveantigen (PA) in a pharmaceutically acceptable carrier, excipient ordiluent.

In one embodiment of a method of treating pain described herein, themethod comprises administering to a subject in need thereof, nativemature anthrax toxin protective antigen (PA) and anthrax toxin edemafactor (EF), anthrax toxin lethal factor (LF) or any combinationthereof.

In one embodiment of an aspect described herein in which PA is part ofthe fusion protein or part of the composition that is administered forpain treatment, the PA or mPA is administered in an oligomeric form. Inone embodiment, the oligomeric PA or mPA is formed from proteolyticallyactivated PA or mPA (or mutant thereof) to achieve increased avidity forreceptor-bearing cells. By way of non-limiting example, PA can betreated with trypsin to nick and then separate the two fragments (e.g.PA63 and PA20) on an ion exchange column. PA63 will elute as an oligomer(e.g. a heptamer) and will remain in the proteolytically activatedprepore state if the pH is kept above about pH 8.0. Preparation ofoligomeric and/or proteolytically activated PA is described in the art,e.g., in Milne et al, JBC 269: 20607-20612, 1994; which is incorporatedby reference herein in its entirety.

In one embodiment of an aspect involving administering a first andsecond composition, the first composition comprises both i) an anthraxtoxin protective antigen (PA) moiety and ii) a mutant anthrax toxinprotective antigen (mPA) moiety that has been altered to block itsnative receptor-binding function, fused with a molecule capable ofspecifically targeting a nociceptor surface receptor or an ion channelreceptor. In another embodiment including administration of a firstcomposition comprising i) anthrax toxin protective antigen (PA) moietyand ii) mutant anthrax toxin protective antigen (mPA) moiety that hasbeen altered to block its native receptor-binding function, fused with amolecule capable of specifically targeting a nociceptor surface receptoror an ion channel receptor, the two different first polypeptides are inoligomeric form such that the oligomeric complex comprises bothpolypeptides. In one embodiment, the first composition is administeredin a separate injection before, simultaneously or after administeringthe second composition.

The compositions and methods described herein can be administered to asubject having or diagnosed as having pain. Thus, the methods describedherein encompass administering an effective amount of a compositiondescribed herein to a subject in order to alleviate pain. As usedherein, “alleviating pain” is ameliorating any condition or symptomassociated with the pain. As compared with an equivalent untreatedcontrol, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%,90%, 95%, 99% or more as measured by any standard technique. A varietyof means for administering the compositions described herein to subjectsare known to those of skill in the art. Such methods can include, butare not limited to oral, parenteral, intravenous, intramuscular,subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous,topical, injection, or intratumoral administration. Administration canbe local or systemic.

The term “effective amount” as used herein refers to the amount of acomposition needed to alleviate at least one or more symptom of thedisease or disorder, and relates to a sufficient amount ofpharmacological composition to provide the desired effect. The term“therapeutically effective amount” therefore refers to an amount of thecomposition that is sufficient to provide a particular anti-pain effectwhen administered to a typical subject. An effective amount as usedherein, in various contexts, would also include an amount sufficient todelay the development of a symptom of the disease, alter the course of asymptom disease (for example but not limited to, slowing the progressionof a symptom of the disease), or reverse a symptom of the disease. Thus,it is not generally practicable to specify an exact “effective amount”.However, for any given case, an appropriate “effective amount” can bedetermined by one of ordinary skill in the art using only routineexperimentation.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dosage can vary depending upon the dosage formemployed and the route of administration utilized. The dose ratiobetween toxic and therapeutic effects is the therapeutic index and canbe expressed as the ratio LD50/ED50. Compositions and methods thatexhibit large therapeutic indices are preferred. A therapeuticallyeffective dose can be estimated initially from cell culture assays.Also, a dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC50 (i.e., theconcentration of the active ingredient which achieves a half-maximalinhibition of symptoms) as determined in cell culture, or in anappropriate animal model. Levels in plasma can be measured, for example,by high performance liquid chromatography. The effects of any particulardosage can be monitored by a suitable bioassay. The dosage can bedetermined by a physician and adjusted, as necessary, to suit observedeffects of the treatment.

In some embodiments of all the aspects described herein, the technologydescribed herein relates to a pharmaceutical composition as describedherein, and optionally combined with a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers and diluents includesaline, aqueous buffer solutions, solvents and/or dispersion media. Theuse of such carriers and diluents is well known in the art. Somenon-limiting examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” or the like are used interchangeably herein. In someembodiments of all the aspects described herein, the carrier inhibitsthe degradation of the active agent, e.g. a composition as describedherein.

In one embodiment of any aspect described herein involving apharmaceutical composition, the pharmaceutical composition can be aparenteral dose form. Since administration of parenteral dosage formstypically bypasses the patient's natural defenses against contaminants,parenteral dosage forms are preferably sterile or capable of beingsterilized prior to administration to a patient. Examples of parenteraldosage forms include, but are not limited to, solutions ready forinjection, dry products ready to be dissolved or suspended in apharmaceutically acceptable vehicle for injection, suspensions ready forinjection, and emulsions. In addition, controlled-release parenteraldosage forms can be prepared for administration of a patient, including,but not limited to, DUROS®-type dosage forms and dose-dumping.

Suitable vehicles that can be used to provide parenteral dosage formsare well known to those skilled in the art. Examples include, withoutlimitation: sterile water; water for injection USP; saline solution;glucose solution; aqueous vehicles such as but not limited to, sodiumchloride injection, Ringer's injection, dextrose Injection, dextrose andsodium chloride injection, and lactated Ringer's injection;water-miscible vehicles such as, but not limited to, ethyl alcohol,polyethylene glycol, and propylene glycol; and non-aqueous vehicles suchas, but not limited to, corn oil, cottonseed oil, peanut oil, sesameoil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compoundsthat alter or modify the solubility of a pharmaceutically acceptablesalt of a composition as disclosed herein can also be incorporated intothe parenteral dosage forms of the disclosure, including conventionaland controlled-release parenteral dosage forms.

Conventional dosage forms generally provide rapid or immediate drugrelease from the formulation. Depending on the pharmacology andpharmacokinetics of the drug, use of conventional dosage forms can leadto wide fluctuations in the concentrations of the drug in a patient'sblood and other tissues. These fluctuations can impact a number ofparameters, such as dose frequency, onset of action, duration ofefficacy, maintenance of therapeutic blood levels, toxicity, sideeffects, and the like. Advantageously, controlled-release formulationscan be used to control a drug's onset of action, duration of action,plasma levels within the therapeutic window, and peak blood levels. Inparticular, controlled- or extended-release dosage forms or formulationscan be used to ensure that the maximum effectiveness of a drug isachieved while minimizing potential adverse effects and safety concerns,which can occur both from under-dosing a drug (i.e., going below theminimum therapeutic levels) as well as exceeding the toxicity level forthe drug. In some embodiments of all the aspects described herein, thecomposition can be administered in a sustained release formulation.

Controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledrelease counterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include: 1) extended activity of the drug; 2) reduceddosage frequency; 3) increased patient compliance; 4) usage of lesstotal drug; 5) reduction in local or systemic side effects; 6)minimization of drug accumulation; 7) reduction in blood levelfluctuations; 8) improvement in efficacy of treatment; 9) reduction ofpotentiation or loss of drug activity; and 10) improvement in speed ofcontrol of diseases or conditions. Kim, Cherng-ju, Controlled ReleaseDosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release other amountsof drug to maintain this level of therapeutic or prophylactic effectover an extended period of time. In order to maintain this constantlevel of drug in the body, the drug must be released from the dosageform at a rate that will replace the amount of drug being metabolizedand excreted from the body. Controlled-release of an active ingredientcan be stimulated by various conditions including, but not limited to,pH, ionic strength, osmotic pressure, temperature, enzymes, water, andother physiological conditions or compounds.

A variety of known controlled- or extended-release dosage forms,formulations, and devices can be adapted for use with the salts andcompositions of the disclosure. Examples include, but are not limitedto, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809;3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548;5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B 1; each ofwhich is incorporated herein by reference. These dosage forms can beused to provide slow or controlled-release of one or more activeingredients using, for example, hydroxypropylmethyl cellulose, otherpolymer matrices, gels, permeable membranes, osmotic systems (such asOROS® (Alza Corporation, Mountain View, Calif. USA)), or a combinationthereof to provide the desired release profile in varying proportions.

The methods described herein can further comprise administering a secondagent and/or treatment to the subject, e.g. as part of a combinatorialtherapy. Non-limiting examples of a second agent and/or treatment caninclude pain relievers, anti-inflammatories, and other medications thattreat pain and/or a condition causing pain.

In one embodiment of any aspect described herein involving theadministration of an effective dose of a composition, an effective doseof a composition as described herein can be administered to a patientonce. In another embodiment, an effective dose of a composition can beadministered to a patient repeatedly. For systemic administration,subjects can be administered a therapeutic amount of a composition asdescribed herein, such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30mg/kg, 40 mg/kg, 50 mg/kg, or more.

In one embodiments of any of the aspects described herein involvingadministration of a composition for the treatment of pain, after aninitial treatment regimen, the treatments can be administered on a lessfrequent basis. For example, after treatment biweekly for three months,treatment can be repeated once per month, for six months or a year orlonger. Treatment according to the methods described herein can reducelevels of a marker or symptom of a condition, e.g. pain by at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80% or at least 90% ormore.

The dosage of a composition as described herein can be determined by aphysician and adjusted, as necessary, to suit observed effects of thetreatment. With respect to duration and frequency of treatment, it istypical for skilled clinicians to monitor subjects in order to determinewhen the treatment is providing therapeutic benefit, and to determinewhether to increase or decrease dosage, increase or decreaseadministration frequency, discontinue treatment, resume treatment, ormake other alterations to the treatment regimen. The dosing schedule canvary from once a week to daily depending on a number of clinicalfactors, such as the subject's sensitivity to the composition. Thedesired dose or amount of activation can be administered at one time ordivided into subdoses, e.g., 2-4 subdoses and administered over a periodof time, e.g., at appropriate intervals through the day or otherappropriate schedule. In some embodiments of all the aspects describedherein, administration can be chronic, e.g., one or more doses and/ortreatments daily over a period of weeks or months. Examples of dosingand/or treatment schedules are administration daily, twice daily, threetimes daily or four or more times daily over a period of 1 week, 2weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5months, or 6 months, or more. A composition can be administered over aperiod of time, such as over a 5 minute, 10 minute, 15 minute, 20minute, or 25 minute period.

The dosage ranges for the administration of a composition as describedherein, according to the methods described herein depend upon, forexample, the form of the active ingredient, its potency, and the extentto which symptoms, markers, or indicators of a condition describedherein are desired to be reduced, for example the percentage reductiondesired for pain. The dosage should not be so large as to cause adverseside effects. Generally, the dosage will vary with the age, condition,and sex of the patient and can be determined by one of skill in the art.The dosage can also be adjusted by the individual physician in the eventof any complication.

The efficacy of a composition in, e.g. the treatment of a conditiondescribed herein, or to induce a response as described herein (e.g. areduction of pain) can be determined by the skilled clinician. However,a treatment is considered “effective treatment,” as the term is usedherein, if one or more of the signs or symptoms of a condition describedherein are altered in a beneficial manner, other clinically acceptedsymptoms are improved, or even ameliorated, or a desired response isinduced e.g., by at least 10% following treatment according to themethods described herein. Efficacy can be assessed, for example, bymeasuring a marker, indicator, symptom, and/or the incidence of acondition treated according to the methods described herein or any othermeasurable parameter appropriate. Efficacy can also be measured by afailure of an individual to worsen as assessed by hospitalization, orneed for medical interventions (i.e., progression of the disease ishalted). Methods of measuring these indicators are known to those ofskill in the art and/or are described herein. Treatment includes anytreatment of a disease in an individual or an animal (some non-limitingexamples include a human or an animal) and includes: (1) inhibiting thedisease, e.g., preventing a worsening of symptoms (e.g. pain orinflammation); or (2) relieving the severity of the disease, e.g.,causing regression of symptoms. An effective amount for the treatment ofa disease means that amount which, when administered to a subject inneed thereof, is sufficient to result in effective treatment as thatterm is defined herein, for that disease. Efficacy of an agent can bedetermined by assessing physical indicators of a condition or desiredresponse. It is well within the ability of one skilled in the art tomonitor efficacy of administration and/or treatment by measuring any oneof such parameters, or any combination of parameters. Efficacy can beassessed in animal models of a condition described herein, for exampletreatment of a mouse model of pain. When using an experimental animalmodel, efficacy of treatment is evidenced when a statisticallysignificant change in a marker is observed, e.g. responses to oravoidance of stimuli in the affected area.

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed invention, because the scope of theinvention is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. If there is an apparent discrepancy between the usageof a term in the art and its definition provided herein, the definitionprovided within the specification shall prevail.

Definitions

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here.

As used herein, the term “capable of” when used with a verb, encompassesor means the action of the corresponding verb. For example, “capable ofblocking” also means blocks, “capable of cleaving” also means cleaves,“capable of binding” also means binds, “capable of translocating” alsomeans translocates, and “capable of specifically targeting . . . ” alsomeans specifically targets.

As used herein, a “nociceptor neuron-binding protein” or“nociceptor-binding protein” when used in reference with a fusionprotein described herein refers to a polypeptide targeting moiety (TM)that is capable of binding to a binding site on the nociceptor neuron,wherein the interaction results in that binding site of the neuronundergoing endocytosis to be incorporated into an endosome within thenociceptor neuron. In one embodiment, the nociceptor neuron-bindingprotein is an antibody or antibody fragment thereof that binds areceptor or ion channel expressed on the cell surface of the nociceptorneuron, e.g., the nerve grow factor receptor or the ANTXR2 or Nav1.7,Nav1.8, and Nav1.9 ion channel proteins. In one embodiment, thenociceptor neuron-binding protein is a ligand for a cell surfacereceptor of the nociceptor neuron, e.g., the nerve grow factor ligandfor the nerve grow factor receptor. In one embodiment, the nociceptorneuron-binding protein is a PA or a variant form of PA or PA fragmentsthereof that is capable of binding to its receptor, ANTXR2. In oneembodiment, the TM targets binds to the ANTXR2 (CMG2) receptor expressedon the nociceptor neuron.

In one embodiment, a variant form of PA that is capable of binding orbinds to its receptor is resistant to furin protease and furin-likeproteases. In one embodiment, a variant form of PA is modified (e.g.,chemically) or mutated at the furin cleavage site. In some embodiments,the PA^(furin-) is mutated at the furin cleavage site ¹⁶⁴RKKR¹⁶⁷ toamino acid residues SSSR (SEQ ID NO: 32) or SSSS (SEQ ID NO: 33). Theamino acid numbering is with reference to a PA polypeptide without the29 residue signal peptide at the N-terminus. In one embodiment, PAfragments thereof that is capable of binding to its receptor are PA63,the fragment produced by furin cleavage of the full-length PA protein,and PAd4, the C-terminal receptor binding part of the full-length nativePA. In one embodiment, the PA fragments thereof that is capable ofbinding to its receptor is PAd4 plus at least 1-60 consecutive aminoacid residues N-terminal to PAd4 domain in the native PA, meaning theestimated PAd4 sequence plus additional upstream sequence is theC-terminal receptor binding section of PA.

The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all usedherein to mean a decrease by a statistically significant amount. In someembodiments, “reduce,” “reduction” or “decrease” or “inhibit” typicallymeans a decrease by at least 10% as compared to a reference level (e.g.the absence of a given treatment) and can include, for example, adecrease by at least about 10%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or more. As used herein,“reduction” or “inhibition” does not encompass a complete inhibition orreduction as compared to a reference level. “Complete inhibition” is a100% inhibition as compared to a reference level. A decrease can bepreferably down to a level accepted as within the range of normal for anindividual without a given disorder.

The terms “increased”, “increase”, “enhance”, or “activate” are all usedherein to mean an increase by a statically significant amount. In someembodiments of all the aspects described herein, the terms “increased”,“increase”, “enhance”, or “activate” can mean an increase of at least10% as compared to a reference level, for example an increase of atleast about 20%, or at least about 30%, or at least about 40%, or atleast about 50%, or at least about 60%, or at least about 70%, or atleast about 80%, or at least about 90% or up to and including a 100%increase or any increase between 10-100% as compared to a referencelevel, or at least about a 2-fold, or at least about a 3-fold, or atleast about a 4-fold, or at least about a 5-fold or at least about a10-fold increase, or any increase between 2-fold and 10-fold or greateras compared to a reference level. In the context of a marker or symptom,an “increase” is a statistically significant increase in such level.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Insome embodiments of all the aspects described herein, the subject is amammal, e.g., a primate, e.g., a human. The terms, “individual,”“patient” and “subject” are used interchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but is notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models of pain. Asubject can be male or female.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a condition in need of treatment(e.g. pain) or one or more complications related to such a condition,and optionally, have already undergone treatment for pain or the one ormore complications related to pain. Alternatively, a subject can also beone who has not been previously diagnosed as having pain or one or morecomplications related to pain. For example, a subject can be one whoexhibits one or more risk factors for pain or one or more complicationsrelated to pain or a subject who does not exhibit risk factors.

A “subject in need” of treatment for a particular condition can be asubject having that condition, diagnosed as having that condition, or atrisk of developing that condition.

As used herein, “engineered” refers to the aspect of having beenmanipulated by the hand of man. For example, an fusion polypeptide isconsidered to be “engineered” when the sequence of the polypeptideand/or encoding nucleic acid sequence manipulated by the hand of man todiffer from the sequence of a polypeptide as it exists in nature. As iscommon practice and is understood by those in the art, progeny andcopies of an engineered polynucleotide and/or polypeptide are typicallystill referred to as “engineered” even though the actual manipulationwas performed on a prior entity.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably herein to designate a series of amino acid residues,connected to each other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and“polypeptide” refer to a polymer of amino acids, including modifiedamino acids (e.g., phosphorylated, glycated, glycosylated, etc.) andamino acid analogs, regardless of its size or function. “Protein” and“polypeptide” are often used in reference to relatively largepolypeptides, whereas the term “peptide” is often used in reference tosmall polypeptides, but usage of these terms in the art overlaps. Theterms “protein” and “polypeptide” are used interchangeably herein whenreferring to a gene product and fragments thereof. Thus, exemplarypolypeptides or proteins include gene products, naturally occurringproteins, homologs, orthologs, paralogs, fragments and otherequivalents, variants, fragments, and analogs of the foregoing.

In some embodiments of all the aspects described herein, a polypeptide,e.g., a fusion polypeptide or portion thereof (e.g. a domain), can be avariant of a sequence described herein. In some embodiments of all theaspects described herein, the variant is a conservative substitutionvariant. A “variant,” as referred to herein, is a polypeptidesubstantially homologous to a native or reference polypeptide, but whichhas an amino acid sequence different from that of the native orreference polypeptide because of one or a plurality of deletions,insertions or substitutions. Polypeptide-encoding DNA sequencesencompass sequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to a native or reference DNAsequence, but that encode a variant protein or fragment thereof thatretains the relevant biological activity relative to the referenceprotein, e.g., at least 50% of the wildtype reference protein. As toamino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters a single amino acid or asmall percentage, (i.e. 5% or fewer, e.g. 4% or fewer, or 3% or fewer,or 1% or fewer) of amino acids in the encoded sequence is a“conservatively modified variant” where the alteration results in thesubstitution of an amino acid with a chemically similar amino acid. Itis contemplated that some changes can potentially improve the relevantactivity, such that a variant, whether conservative or not, has morethan 100% of the activity of wildtype, e.g. 110%, 125%, 150%, 175%,200%, 500%, 1000% or more.

A given amino acid can be replaced by a residue having similarphysiochemical characteristics, e.g., substituting one aliphatic residuefor another (such as Ile, Val, Leu, or Ala for one another), orsubstitution of one polar residue for another (such as between Lys andArg; Glu and Asp; or Gln and Asn). Other such conservativesubstitutions, e.g., substitutions of entire regions having similarhydrophobicity characteristics, are known. Polypeptides comprisingconservative amino acid substitutions can be tested in any one of theassays described herein to confirm that a desired activity of a nativeor reference polypeptide is retained. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and allelesconsistent with the disclosure. Typically conservative substitutions forone another include: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D),Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R),Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,Creighton, Proteins (1984)).

Any cysteine residue not involved in maintaining the proper conformationof the polypeptide also can be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) can be added to thepolypeptide to improve its stability or facilitate oligomerization.

In one embodiment of any the aspects described herein involvingadministering a polypeptide, the polypeptide administered to a subjectcan comprise one or more amino acid substitutions or modifications. Inone embodiment, the substitutions and/or modifications can prevent orreduce proteolytic degradation and/or prolong half-life of thepolypeptide in the subject. In one embodiment, a polypeptide can bemodified by conjugating or fusing it to other polypeptide or polypeptidedomains such as, by way of non-limiting example, transferrin(WO06096515A2), albumin (Yeh et al., 1992), growth hormone(US2003104578AA); cellulose (Levy and Shoseyov, 2002); and/or Fcfragments (Ashkenazi and Chamow, 1997). The references in the foregoingparagraph are incorporated by reference herein in their entireties.

In one embodiment of any of the aspects described herein involving apolypeptide, a polypeptide as described herein can comprise at least onepeptide bond replacement. A single peptide bond or multiple peptidebonds, e.g. 2 bonds, 3 bonds, 4 bonds, 5 bonds, or 6 or more bonds, orall the peptide bonds can be replaced. An isolated peptide as describedherein can comprise one type of peptide bond replacement or multipletypes of peptide bond replacements, e.g. 2 types, 3 types, 4 types, 5types, or more types of peptide bond replacements. Non-limiting examplesof peptide bond replacements include urea, thiourea, carbamate, sulfonylurea, trifluoroethylamine, ortho-(aminoalkyl)-phenylacetic acid,para-(aminoalkyl)-phenylacetic acid, meta-(aminoalkyl)-phenylaceticacid, thioamide, tetrazole, boronic ester, olefinic group, andderivatives thereof.

In one embodiment of any of the aspects described herein involving apolypeptide, a polypeptide as described herein can comprise naturallyoccurring amino acids commonly found in polypeptides and/or proteinsproduced by living organisms, e.g. Ala (A), Val (V), Leu (L), Ile (I),Pro (P), Phe (F), Trp (W), Met (M), Gly (G), Ser (S), Thr (T), Cys (C),Tyr (Y), Asn (N), Gln (Q), Asp (D), Glu (E), Lys (K), Arg (R), and His(H). In one embodiment of any of the aspects described herein involvinga polypeptide, a polypeptide as described herein can comprisealternative amino acids. Non-limiting examples of alternative aminoacids include D-amino acids, beta-amino acids, homocysteine,phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline,gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylicacid, statine, 1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid,penicillamine (3-mercapto-D-valine), ornithine, citruline,alpha-methyl-alanine, para-benzoylphenylalanine, para-aminophenylalanine, p-fluorophenylalanine, phenylglycine, propargylglycine,sarcosine, and tert-butylglycine), diaminobutyric acid,7-hydroxy-tetrahydroisoquinoline carboxylic acid, naphthylalanine,biphenylalanine, cyclohexylalanine, amino-isobutyric acid, norvaline,norleucine, tert-leucine, tetrahydroisoquinoline carboxylic acid,pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine,dehydroleucine, 2,2-diethylglycine, 1-amino-l-cyclopentanecarboxylicacid, 1-amino-l-cyclohexanecarboxylic acid, amino-benzoic acid,amino-naphthoic acid, gamma-aminobutyric acid, difluorophenylalanine,nipecotic acid, alpha-amino butyric acid, thienyl-alanine,t-butylglycine, trifluorovaline; hexafluoroleucine; fluorinated analogs;azide-modified amino acids; alkyne-modified amino acids; cyano-modifiedamino acids; and derivatives thereof.

In one embodiment of any of the aspects described herein involving apolypeptide, a polypeptide can be modified, e.g. by addition of a moietyto one or more of the amino acids comprising the peptide. In oneembodiment, a polypeptide as described herein can comprise one or moremoiety molecules, e.g. 1 or more moiety molecules per peptide, 2 or moremoiety molecules per peptide, 5 or more moiety molecules per peptide, 10or more moiety molecules per peptide or more moiety molecules perpeptide. In some embodiments of all the aspects described herein, apolypeptide as described herein can comprise one more types ofmodifications and/or moieties, e.g. 1 type of modification, 2 types ofmodifications, 3 types of modifications or more types of modifications.Non-limiting examples of modifications and/or moieties includePEGylation; glycosylation; HESylation; ELPylation; lipidation;acetylation; amidation; end-capping modifications; cyano groups;phosphorylation; albumin, and cyclization. In some embodiments of allthe aspects described herein, an end-capping modification can compriseacetylation at the N-terminus, N-terminal acylation, and N-terminalformylation. In some embodiments of all the aspects described herein, anend-capping modification can comprise amidation at the C-terminus,introduction of C-terminal alcohol, aldehyde, ester, and thioestermoieties. The half-life of a polypeptide can be increased by theaddition of moieties, e.g. PEG or albumin.

In one embodiment of any of the aspects described herein involvingadministering a polypeptide (or administering a nucleic acid encoding apolypeptide), the polypeptide administered or encoded can be afunctional fragment of one of the amino acid sequences described herein.As used herein, a “functional fragment” is a fragment or segment of apeptide which retains at least 50% of the wild-type referencepolypeptide's activity according to the assays described below herein. Afunctional fragment can comprise conservative or non-conservativesubstitutions of the sequences disclosed herein.

Alterations of the original amino acid sequence can be accomplished byany of a number of techniques known to one of skill in the art. Aminoacid substitutions can be introduced, for example, at particularlocations by synthesizing oligonucleotides containing a codon change inthe nucleotide sequence encoding the amino acid to be changed, flankedby restriction sites permitting ligation to fragments of the originalsequence. Following ligation, the resulting reconstructed sequenceencodes an analog having the desired amino acid insertion, substitution,or deletion. Alternatively, oligonucleotide-directed site-specificmutagenesis procedures can be employed to provide an altered nucleotidesequence having particular codons altered according to the substitution,deletion, or insertion required. Techniques for making such alterationsinclude those disclosed by Walder et al. (Gene 42:133, 1986); Bauer etal. (Gene 37:73, 1985); Craik (BioTechniques, Jan. 1985, 12-19); Smithet al. (Genetic Engineering: Principles and Methods, Plenum Press,1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are hereinincorporated by reference in their entireties. In some embodiments ofall the aspects described herein, a polypeptide as described herein canbe chemically synthesized and mutations can be incorporated as part ofthe chemical synthesis process.

As used herein an “antibody” refers to IgG, IgM, IgA, IgD or IgEmolecules or antigen-specific antibody fragments thereof (including, butnot limited to, a Fab, F(ab′)2, Fv, disulfide linked Fv, scFv, singledomain antibody, closed conformation multispecific antibody,disulfide-linked scfv, diabody), whether derived from any species thatnaturally produces an antibody, or created by recombinant DNAtechnology; whether isolated from serum, B-cells, hybridomas,transfectomas, yeast or bacteria.

As described herein, an “antigen” is a molecule that is bound by abinding site on an antibody agent. Typically, antigens are bound byantibody ligands and are capable of raising an antibody response invivo. An antigen can be a polypeptide, protein, nucleic acid or othermolecule or portion thereof. The term “antigenic determinant” refers toan epitope on the antigen recognized by an antigen-binding molecule, andmore particularly, by the antigen-binding site of the molecule.

As used herein, the term “antibody reagent” refers to a polypeptide thatincludes at least one immunoglobulin variable domain or immunoglobulinvariable domain sequence and which specifically binds a given antigen.An antibody reagent can comprise an antibody or a polypeptide comprisingan antigen-binding domain of an antibody. In some embodiments of all theaspects described herein, an antibody reagent can comprise a monoclonalantibody or a polypeptide comprising an antigen-binding domain of amonoclonal antibody. For example, an antibody can include a heavy (H)chain variable region (abbreviated herein as VH), and a light (L) chainvariable region (abbreviated herein as VL). In another example, anantibody includes two heavy (H) chain variable regions and two light (L)chain variable regions. The term “antibody reagent” encompassesantigen-binding fragments of antibodies (e.g., single chain antibodies,Fab and sFab fragments, F(ab′)2, Fd fragments, Fv fragments, scFv, anddomain antibodies (dAb) fragments (see, e.g. de Wildt et al., Eur J.Immunol. 1996; 26(3):629-39; which is incorporated by reference hereinin its entirety)) as well as complete antibodies. An antibody can havethe structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypesand combinations thereof). Antibodies can be from any source, includingmouse, rabbit, pig, rat, and primate (human and non-human primate) andprimatized antibodies. Antibodies also include midibodies, humanizedantibodies, chimeric antibodies, and the like.

The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed “frameworkregions” (“FR”). The extent of the framework region and CDRs has beenprecisely defined (see, Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, and Chothia, C.et al. (1987) J. Mol. Biol. 196:901-917; which are incorporated byreference herein in their entireties). Each VH and VL is typicallycomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The terms “antigen-binding fragment” or “antigen-binding domain”, whichare used interchangeably herein are used to refer to one or morefragments of a full length antibody that retain the ability tospecifically bind to a target of interest. Examples of binding fragmentsencompassed within the term “antigen-binding fragment” of a full lengthantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment including two Fab fragments linked by a disulfide bridge at thehinge region; (iii) an Fd fragment consisting of the VH and CH1 domains;(iv) an Fv fragment consisting of the VL and VH domains of a single armof an antibody, (v) a dAb fragment (Ward et al., (1989) Nature341:544-546; which is incorporated by reference herein in its entirety),which consists of a VH or VL domain; and (vi) an isolatedcomplementarity determining region (CDR) that retains specificantigen-binding functionality. As used herein, the term “specificbinding” refers to a chemical interaction between two molecules,compounds, cells and/or particles wherein the first entity binds to thesecond, target entity with greater specificity and affinity than itbinds to a third entity which is a non-target. In some embodiments ofall the aspects described herein, specific binding can refer to anaffinity of the first entity for the second target entity which is atleast 10 times, at least 50 times, at least 100 times, at least 500times, at least 1000 times or greater than the affinity for the thirdnontarget entity.

Additionally, and as described herein, an antibody can be furtheroptimized to decrease potential immunogenicity, while maintainingfunctional activity, for therapy in humans. In this regard, functionalactivity means a polypeptide capable of displaying one or more knownfunctional activities associated with a recombinant antibody or antibodyreagent thereof as described herein. Such functional activities include,e.g. the ability to bind to the target molecule.

As used herein, the term “nucleic acid” or “nucleic acid sequence”refers to any molecule, preferably a polymeric molecule, incorporatingunits of ribonucleic acid, deoxyribonucleic acid or an analog thereof.The nucleic acid can be either single-stranded or double-stranded. Asingle-stranded nucleic acid can be one nucleic acid strand of adenatured double-stranded DNA. Alternatively, it can be asingle-stranded nucleic acid not derived from any double-stranded DNA.In one aspect, the nucleic acid can be DNA. In another aspect, thenucleic acid can be RNA. Suitable nucleic acid molecules are DNA,including genomic DNA or cDNA. Other suitable nucleic acid molecules areRNA, including mRNA.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with a disease ordisorder, e.g. pain. The term “treating” includes reducing oralleviating at least one adverse effect or symptom of a condition,disease or disorder associated with pain. Treatment is generally“effective” if one or more symptoms or clinical markers are reduced.Alternatively, treatment is “effective” if the progression of a diseaseis reduced or halted. That is, “treatment” includes not just theimprovement of symptoms or markers, but also a cessation of, or at leastslowing of, progress or worsening of symptoms compared to what would beexpected in the absence of treatment. Beneficial or desired clinicalresults include, but are not limited to, alleviation of one or moresymptom(s), diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, remission (whetherpartial or total), and/or decreased mortality, whether detectable orundetectable. The term “treatment” of a disease also includes providingrelief from the symptoms or side-effects of the disease (includingpalliative treatment).

As used herein, the term “pharmaceutical composition” refers to theactive agent in combination with a pharmaceutically acceptable carriere.g. a carrier commonly used in the pharmaceutical industry. The phrase“pharmaceutically acceptable” is employed herein to refer to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used herein, the term “administering,” refers to the placement of acompound as disclosed herein into a subject by a method or route whichresults in at least partial delivery of the agent at a desired site.Pharmaceutical compositions comprising the compounds disclosed hereincan be administered by any appropriate route which results in aneffective treatment in the subject.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean ±1%.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the method or composition, yet open to the inclusion ofunspecified elements, whether essential or not.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

As used herein, a protease activity included in a fusion proteinembraces all non-cytotoxic proteases that are capable of cleaving one ormore proteins of the exocytic fusion apparatus in eukaryotic cells. Theprotease is preferably a bacterial protease (or fragment thereof). Morepreferably the bacterial protease is selected from the generaClostridium or Neisseria/Streptococcus (e.g. a clostridial L-chain, or aneisserial IgA protease preferably from N. gonorrhoeae or S.pneumoniae). Another example of non-cytotoxic protease includes scorpionvenom protease, such as those from the venom of the Brazilian scorpionTityus serrulatus, or the protease antarease.

Protease activities also embrace the activities of variant non-cytotoxicproteases (i.e. variants of naturally-occurring protease molecules), solong as the variant proteases still demonstrate the requisite proteaseactivity. By way of example, a variant may have at least 70%, preferablyat least 80%, more preferably at least 90%, and most preferably at least95% or at least 98% amino acid sequence homology with a referenceprotease sequence. Thus, the term variant includes non-cytotoxicproteases having enhanced (or decreased) endopeptidaseactivity—particular mention here is made to the increased Kcat/Km ofBTx/A mutants Q161A, E54A, and K165L see Ahmed, S. A. (2008) Protein J.DOI 10.1007/s10930-007-9118-8, which is incorporated by referencethereto. The term fragment, when used in relation to a protease,typically means a peptide having at least 150, preferably at least 200,more preferably at least 250, and most preferably at least 300 aminoacid residues of the reference protease. As with the TM ‘fragment’component (discussed above), protease ‘fragments’ of the presentinvention embrace fragments of variant proteases based on a referencesequence.

In one embodiment of any of the aspects described herein, the proteaseactivity included in a fusion protein demonstrates a serine ormetalloprotease activity (e.g. endopeptidase activity). In oneembodiment, the protease is specific for a SNARE protein (e.g. SNAP-25,synaptobrevin/VAMP, or syntaxin).

Particular mention is made to the protease domains of neurotoxins, forexample the protease domains of bacterial neurotoxins. Thus, the variousaspects described herein embrace the use of neurotoxin domains whichoccur in nature, as well as recombinantly prepared versions of suchnaturally-occurring neurotoxins.

Exemplary neurotoxins are produced by clostridia, and the termclostridial neurotoxin embraces neurotoxins produced by C. tetani (TTx),and by C. botulinum (BTx) serotypes A-G, as well as the closely relatedBTx-like neurotoxins produced by C. baratii and C. butyricum. Theabove-mentioned abbreviations are used throughout the presentspecification. For example, the nomenclature BTx/A denotes the source ofneurotoxin as BTx (serotype A). Corresponding nomenclature applies toother BTx serotypes.

BTxs are the most potent toxins known, with median lethal dose (LD50)values for mice ranging from 0.5 to 5 ng/kg depending on the serotype.BTxs are adsorbed in the gastrointestinal tract, and, after entering thegeneral circulation, bind to the presynaptic membrane of cholinergicnerve terminals and prevent the release of their neurotransmitteracetylcholine. BTx/B, BTx/D, BTx/F and BTx/G cleavesynaptobrevin/vesicle-associated membrane protein (VAMP); BTx/C, BTx/Aand BTx/E cleave the synaptosomal-associated protein of 25 kDa(SNAP-25); and BTx/C cleaves syntaxin.

BTxs share a common structure, being di-chain proteins of ˜150 kDa,consisting of a heavy chain (H-chain) of ˜100 kDa covalently joined by asingle disulfide bond to a light chain (L-chain) of ˜50 kDa. The H-chainconsists of two domains, each of ˜50 kDa. The C-terminal domain (H_(C))is required for the high-affinity neuronal binding, whereas theN-terminal domain (H_(N)) is proposed to be involved in membranetranslocation. The L-chain is a zinc-dependent metalloproteaseresponsible for the cleavage of the substrate SNARE protein.

The term L-chain fragment means a component of the L-chain of aneurotoxin, which fragment demonstrates a metalloprotease activity andis capable of proteolytically cleaving a vesicle and/or plasma membraneassociated protein involved in cellular exocytosis.

Examples of suitable protease (reference) sequences include:

-   Botulinum type A neurotoxin—amino acid residues (1-448)-   Botulinum type B neurotoxin—amino acid residues (1-440)-   Botulinum type C neurotoxin—amino acid residues (1-441)-   Botulinum type D neurotoxin—amino acid residues (1-445)-   Botulinum type E neurotoxin—amino acid residues (1-422)-   Botulinum type F neurotoxin—amino acid residues (1-439)-   Botulinum type G neurotoxin—amino acid residues (1-441)-   Tetanus neurotoxin—amino acid residues (1-457)-   IgA protease—amino acid residues (1-959) Pohlner, J. et al. (1987).    Nature 325, pp. 458-462, which is hereby incorporated by reference    thereto.

In one embodiment of a fusion protein described herein having anon-cytotoxin protease, the non-cytotoxin protease can be an IgAprotease or an Antarease described herein. In one embodiment of a fusionprotein described herein having a non-cytotoxin protease, thenon-cytotoxin protease can have a unique cleavage recognition sequencedescribed in the following pages.

A variety of clostridial toxin fragments comprising the light chain canbe useful in aspects of the present invention with the proviso thatthese light chain fragments can specifically target the core componentsof the neurotransmitter release apparatus and thus participate inexecuting the overall cellular mechanism whereby a clostridial toxinproteolytically cleaves a substrate. The light chains of clostridialtoxins are approximately 420-460 amino acids in length and comprise anenzymatic domain. Research has shown that the entire length of aclostridial toxin light chain is not necessary for the enzymaticactivity of the enzymatic domain. As a non-limiting example, the firsteight amino acids of the BTx/A light chain are not required forenzymatic activity. As another non-limiting example, the first eightamino acids of the TTx light chain are not required for enzymaticactivity. Likewise, the carboxyl-terminus of the light chain is notnecessary for activity. As a non-limiting example, the last 32 aminoacids of the BTx/A light chain (residues 417-448) are not required forenzymatic activity. As another non-limiting example, the last 31 aminoacids of the TTx light chain (residues 427-457) are not required forenzymatic activity. Thus, aspects of this embodiment can includeclostridial toxin light chains comprising an enzymatic domain having alength of, for example, at least 350 amino acids, at least 375 aminoacids, at least 400 amino acids, at least 425 amino acids and at least450 amino acids. Other aspects of this embodiment can includeclostridial toxin light chains comprising an enzymatic domain having alength of, for example, at most 350 amino acids, at most 375 aminoacids, at most 400 amino acids, at most 425 amino acids and at most 450amino acids.

Further examples of suitable non-cytotoxic proteases are described indetail in WO 2007/106115, which is hereby incorporated in its entiretyby reference thereto.

In one embodiment, the non-cytotoxic protease cleaves a non-neuronalSNARE protein such as a SNAP-23 protein. In one embodiment, thenon-cytotoxic protease is a modified botulinum toxin L-chain capable ofcleaving SNAP-23. An example of such a modified L-chain is described byChen and Barbieri, PNAS, vol. 106, no. 23, p 9180-9184, 2009.

In one embodiment, the non-cytotoxic protease is a BTx/A, BTx/C or BTx/Eprotease, and the preferred SNARE motif is a SNAP (e.g. SNAP 25) motif.

In another embodiment, the non-cytotoxic protease is a BTx/B, BTx/D,BTx/F or BTx/G or tetanus neurotoxin (TTx) protease, and the preferredSNARE motif is a VAMP motif.

In another embodiment, the non-cytotoxic protease is a BTx/C1 protease,and the preferred SNARE motif is a syntaxin motif.

The non-cytotoxic proteases of the engineered fusion proteins describedherein recognise different cleavage site sequences and thus haveslightly different cleavage specificities.

Non- Cleavage site recognition sequence: cytotoxicP4-P3-P2-P1-↓-P1′-P2′-P3′ Protease P4 P3 P2 P1 P1′ P2′ P3′ BTx/A E A N QR A T (SEQ ID NO: 71) BTx/B G A S Q F E T (SEQ ID NO: 72) BTx/C A N Q RA T K (SEQ ID NO: 73) BTx/C D T K K A V K (SEQ ID NO: 74) BTx/D R D Q KL S E (SEQ ID NO: 75) BTx/E Q I D R I M E (SEQ ID NO: 76) BTx/F E R D QK L S (SEQ ID NO: 77) BTx/G E T S A A K I (SEQ ID NO: 78) TTx G A S Q FE T (SEQ ID NO: 79) IgA  S T P P T P S (SEQ ID NO: 80) proteaseAntarease I K R K  Y W W (SEQ ID NO: 81)

By way of further example, reference is made to the followingrecognition sequences and cleavage sites:

Non- Cleavage site recognition sequence: cytotoxicP4-P3-P2-P1-↓-P1′-P2′-P3′ Protease P4 P3 P2 P1 P1′ P2′ P3′ BTx/A E A N QR A T (SEQ ID NO: 82) A N Q R A T K (SEQ ID NO: 83) E A N Q R A T(SEQ ID NO: 84) F A N Q R A T (SEQ ID NO: 85) E A N Q R A T(SEQ ID NO: 86) E A N Q R A I (SEQ ID NO: 87) E A N K A T K(SEQ ID NO: 88) E A N K H A T (SEQ ID NO: 89) E A N K H A N(SEQ ID NO: 90) Q R K H BTx/C D E A N Q R A (SEQ ID NO: 91) E A N Q R AT (SEQ ID NO: 92) A N Q R A T K (SEQ ID NO: 93) N Q R A T K M(SEQ ID NO: 94) A N Q R A I K (SEQ ID NO: 95) A N Q R A H Q(SEQ ID NO: 96) D T K K A V K (SEQ ID NO: 97) K T K K A V K(SEQ ID NO: 98) E T K K A I K (SEQ ID NO: 99) E T K R A M K(SEQ ID NO: 100) D T K K A V R (SEQ ID NO: 101) D T K K A L K(SEQ ID NO: 102) D T K K A M K (SEQ ID NO: 103) E S K K A V K(SEQ ID NO: 104) E T K K A M K (SEQ ID NO: 105) E T K K A V K(SEQ ID NO: 106) K A R A BTx/E Q I D R I M E (SEQ ID NO: 107) Q I Q K IT E (SEQ ID NO: 108) Q I D R I V E (SEQ ID NO: 109) Q F D R I M D(SEQ ID NO: 110) Q F D R I M E (SEQ ID NO: 111) Q L D R I H D(SEQ ID NO: 112) Q I D R I M D (SEQ ID NO: 113) Q V D R I Q Q(SEQ ID NO: 114) R I K I BTx/B G A S Q F E T (SEQ ID NO: 115) A G A S QF E (SEQ ID NO: 116) G A S Q F E S (SEQ ID NO: 117) Q A S Q F E S(SEQ ID NO: 118) G A S Q G E T (SEQ ID NO: 119) G A S Q F E Q(SEQ ID NO: 120) Q A S Q F E A (SEQ ID NO: 121) G A S Q F Q Q(SEQ ID NO: 122) G A S Q F E A (SEQ ID NO: 123) Q F BTx/D R D Q K L S E(SEQ ID NO: 124) R D Q K I S E (SEQ ID NO: 125) K D Q K L A E(SEQ ID NO: 126) K L BTx/F E R D Q K L S (SEQ ID NO: 127) V L E R D Q K(SEQ ID NO: 128) E R D Q K I S (SEQ ID NO: 129) E R D Q A L S(SEQ ID NO: 130) E K D Q K L A (SEQ ID NO: 131) Q K BTx/G E S S A A K I(SEQ ID NO: 132) E T S A A K I (SEQ ID NO: 133) E S S A A K L(SEQ ID NO: 134) E T S A A K L (SEQ ID NO: 135) A A TTx G A S Q F E T(SEQ ID NO: 136) G A S Q G E T (SEQ ID NO: 137) G A S Q F E Q(SEQ ID NO: 138) Q A S Q F E A (SEQ ID NO: 139) G A S Q F E S(SEQ ID NO: 140) Q A S Q F E S (SEQ ID NO: 141) G A S Q F Q Q(SEQ ID NO: 142) G A S Q F E A (SEQ ID NO: 143) Q F IgA S T P P T P S(SEQ ID NO: 144) protease Antarease I K R K Y W W (SEQ ID NO: 145)

A targeting moiety (TM) means any chemical structure that functionallyinteracts with a Binding Site to cause a physical association between afusion polypeptide as described herein and the surface of a target cell.In the context of the present invention, the target cell is a nociceptorneuron. The term TM embraces any molecule (i.e. a naturally occurringmolecule, or a chemically/physically modified variant thereof) that iscapable of binding to a Binding Site on the target cell, which BindingSite is capable of internalization (e.g. endosome formation)—alsoreferred to as receptor-mediated endocytosis. The TM may possess anendosomal membrane translocation function, in which case separate TM andTranslocation Domain components need not be present in an agent of thepresent invention. Throughout the preceding description, specific TMshave been described. Reference to the TMs is merely exemplary, and thepresent invention embraces all variants and derivatives thereof, whichretain the basic binding (i.e. targeting) ability of the exemplifiedTMs.

A TM according to the present invention includes antibodies (e.g.antibody fragments) and binding scaffolds; especially commerciallyavailable antibodies/fragments and scaffolds designed for the purpose ofbinding (e.g. specifically) to target cells.

Protein scaffolds represent a new generation of universal bindingframeworks to complement the expanding repertoire of therapeuticmonoclonal antibodies and derivatives such as scFvs, Fab molecules, dAbs(single-domain antibodies), camelids, diabodies and minibodies, each ofwhich may be employed as a TM of the present invention. Scaffold systemscreate or modify known protein recognition domains either throughcreation of novel scaffolds or modification of known protein bindingdomains. Such scaffolds include but are not limited to:

-   -   (i) protein A based scaffolds—affibodies (Nord, K. et al 1997        “Binding proteins selected from combinatorial libraries of an        alpha-helical bacterial receptor domain”. Nat Biotechnol 15,        772-777);    -   (ii) lipocalin based scaffolds—anticalins (Skerra 2008        “Alternative binding proteins: anticalins—harnessing the        structural plasticity of the lipocalin ligand pocket to engineer        novel binding activities”. FEBS J. 275:2677-83);    -   (iii) fibronectin based scaffolds—adnectin (Dineen et al 2008        “The Adnectin CT-322 is a novel VEGF receptor 2 inhibitor that        decreases tumour burden in an orthotropic mouse model of        pancreatic cancer”. BMC Cancer 8:352);    -   (iv) avimers (Silverman et al 2005 “Multivalent avimer proteins        evolved by exon shuffling of a family of human receptor        domains”. Nat Biotechnol 23:1556-61);    -   (v) ankyrin based scaffolds—darpins (Zahnd et al 2006 “Selection        and characterization of Her2 binding-designed ankyrin repeat        proteins”. J Biol Chem. 281:35167-75); and    -   (vi) centyrin scaffolds—based on a protein fold that has        significant structural homology to Ig domains with loops that        are analogous to CDRs. Ig domains are a common module in human        proteins and have been widely applied as alternative scaffold        proteins. Each of the above ‘scaffold’ publications is hereby        incorporated (in its entirety) by reference thereto.

Binding scaffolds can be used to target particular cell types viainteraction with specific cell surface proteins, receptors or other cellsurface epitopes such as sugar groups. Such modified scaffolds can beengineered onto recombinant non-cytotoxic protease based polypeptides ofthe present invention.

The TM of the present invention binds (preferably specifically binds) toa nociceptor neuron target cell in question. The term “specificallybinds” preferably means that a given TM binds to the target cell with abinding affinity (Ka) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ orgreater, more preferably 10⁸ M⁻¹ or greater, and most preferably, 10⁹M⁻¹ or greater. The term “specifically binds” can also mean that a givenTM binds to a given receptor, e.g., ANTXR2 or NGFR, or Nav1.7. 1.8 and1.9 ion channels found on nociceptor, with a binding affinity (Ka) of10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater, more preferably 10⁸M⁻¹ or greater, and most preferably, 10⁹ M−1 or greater.

Reference to TM in the present specification embraces fragments andvariants thereof, which retain the ability to bind to the target cell inquestion. By way of example, a variant may have at least 80%, preferablyat least 90%, more preferably at least 95%, and most preferably at least97 or at least 99% amino acid sequence homology with the reference TM(e.g. any SEQ ID NO presented in the present specification which definesa TM). Thus, a variant may include one or more analogues of an aminoacid (e.g. an unnatural amino acid), or a substituted linkage. Also, byway of example, the term fragment, when used in relation to a TM, meansa peptide having at least ten, preferably at least twenty, morepreferably at least thirty, and most preferably at least forty aminoacid residues of the reference TM. The term fragment also relates to theabove-mentioned variants. Thus, by way of example, a fragment of thepresent invention may comprise a peptide sequence having at least 10,20, 30 or 40 amino acids, wherein the peptide sequence has at least 80%sequence homology over a corresponding peptide sequence (of contiguous)amino acids of the reference peptide.

It is routine to confirm that a TM binds to the selected target cell.For example, a simple radioactive displacement experiment may beemployed in which tissue or cells representative of a target cell inquestion are exposed to labelled (e.g. tritiated) TM in the presence ofan excess of unlabelled TM. In such an experiment, the relativeproportions of non-specific and specific binding may be assessed,thereby allowing confirmation that the TM binds to the target cell.Optionally, the assay may include one or more binding antagonists, andthe assay may further comprise observing a loss of TM binding. Examplesof this type of experiment can be found in Hulme, E. C. (1990),Receptor-binding studies, a brief outline, pp. 303-311, In Receptorbiochemistry, A Practical Approach, Ed. E. C. Hulme, Oxford UniversityPress.

In the context of the present invention, reference to a peptide TMembraces peptide analogues thereof, so long as the analogue binds to thesame receptor as the corresponding ‘reference’ TM.

The fusion proteins (also referred to herein as polypeptides) describedherein may lack a functional HC (heavy chain) or H_(C) domain(C-terminal moiety of the HC) of a clostridial neurotoxin. In oneembodiment, the polypeptides lack the last 50 C-terminal amino acids ofa clostridial neurotoxin holotoxin. In another embodiment, thepolypeptides lack the last 100, 150, 200, 250, or 300 C-terminal aminoacid residues of a clostridial neurotoxin holotoxin. Alternatively, theHC binding activity may be negated/reduced by mutagenesis—by way ofexample, referring to BTx/A for convenience, modification of one or twoamino acid residue mutations (W1266 to L and Y1267 to F) in theganglioside binding pocket causes the HC region to lose its receptorbinding function. Analogous mutations may be made to non-serotype Aclostridial peptide components, e.g. a construct based on botulinum Bwith mutations (W1262 to L and Y1263 to F) or botulinum E (W1224 to Land Y1225 to F). Other mutations to the active site achieve the sameablation of HC receptor binding activity, e.g. Y1267S in botulinum typeA toxin and the corresponding highly conserved residue in the otherclostridial neurotoxins. Details of this and other mutations aredescribed in Rummel et al (2004) (Molecular Microbiol. 51:631-634),which is hereby incorporated by reference thereto.

The HC peptide of a native clostridial neurotoxin comprisesapproximately 400-440 amino acid residues, and consists of twofunctionally distinct domains of approximately 25 kDa each, namely theN-terminal region (commonly referred to as the H_(N) peptide or domain)and the C-terminal region (commonly referred to as the H_(C) peptide ordomain). Moreover, it has been well documented that the C-terminalregion (H_(C)), which constitutes the C-terminal 160-200 amino acidresidues, is responsible for binding of a clostridial neurotoxin to itsnatural cell receptors, namely to nerve terminals at the neuromuscularjunction. Thus, reference throughout this specification to a clostridialheavy-chain lacking a functional heavy chain HC peptide (or domain) suchthat the heavy-chain is incapable of binding to cell surface receptorsto which a native clostridial neurotoxin binds means that theclostridial heavy-chain simply lacks a functional H_(C) peptide. Inother words, the Hc peptide region is either partially or whollydeleted, or otherwise modified (e.g. through conventional chemical orproteolytic treatment) to inactivate its native binding ability fornerve terminals at the neuromuscular junction.

Thus, in one embodiment, a clostridial H_(N) peptide lacks part of aC-terminal peptide portion (H_(C)) of a clostridial neurotoxin and thuslacks the HC binding function of native clostridial neurotoxin. By wayof example, in one embodiment, the C-terminally extended clostridialH_(N) peptide lacks the C-terminal 40 amino acid residues, or theC-terminal 60 amino acid residues, or the C-terminal 80 amino acidresidues, or the C-terminal 100 amino acid residues, or the C-terminal120 amino acid residues, or the C-terminal 140 amino acid residues, orthe C-terminal 150 amino acid residues, or the C-terminal 160 amino acidresidues of a clostridial neurotoxin heavy-chain. In another embodiment,the clostridial H_(N) peptide of the present invention lacks the entireC-terminal peptide portion (H_(C)) of a clostridial neurotoxin and thuslacks the HC binding function of native clostridial neurotoxin. By wayof example, in one embodiment, the clostridial H_(N) peptide lacks theC-terminal 165 amino acid residues, or the C-terminal 170 amino acidresidues, or the C-terminal 175 amino acid residues, or the C-terminal180 amino acid residues, or the C-terminal 185 amino acid residues, orthe C-terminal 190 amino acid residues, or the C-terminal 195 amino acidresidues of a clostridial neurotoxin heavy-chain. By way of furtherexample, the clostridial H_(N) peptide of the present invention lacks aclostridial H_(C) reference sequence selected from the group consistingof:

Botulinum type A neurotoxin—amino acid residues (Y1111-L1296)

Botulinum type B neurotoxin—amino acid residues (Y1098-E1291)

Botulinum type C neurotoxin—amino acid residues (Y1112-E1291)

Botulinum type D neurotoxin—amino acid residues (Y1099-E1276)

Botulinum type E neurotoxin—amino acid residues (Y1086-K1252)

Botulinum type F neurotoxin—amino acid residues (Y1106-E1274)

Botulinum type G neurotoxin—amino acid residues (Y1106-E1297)

Tetanus neurotoxin—amino acid residues (Y1128-D1315).

A Translocation Domain is a molecule or protein domain that enablestranslocation of a protease into a target cell such that a functionalexpression of protease activity occurs within the cytosol of the targetcell. Whether any molecule (e.g. a protein or peptide) possesses therequisite translocation function of the present invention may beconfirmed by any one of a number of conventional assays.

For example, Shone C. (1987) describes an in vitro assay employingliposomes, which are challenged with a test molecule. Presence of therequisite translocation function is confirmed by release from theliposomes of K+ and/or labelled NAD, which may be readily monitored [seeShone C. (1987) Eur. J. Biochem; vol. 167(1): pp. 175-180].

A further example is provided by Blaustein R. (1987), which describes asimple in vitro assay employing planar phospholipid bilayer membranes.The membranes are challenged with a test molecule and the requisitetranslocation function is confirmed by an increase in conductance acrossthe membranes [see Blaustein (1987) FEBS Letts; vol. 226, no. 1: pp.115-120].

Additional methodology to enable assessment of membrane fusion and thusidentification of Translocation Domains suitable for use in the presentinvention are provided by Methods in Enzymology Vol 220 and 221,Membrane Fusion Techniques, Parts A and B, Academic Press 1993.

The present invention also embraces variant translocation domains, solong as the variant domains still demonstrate the requisitetranslocation activity. By way of example, a variant may have at least70%, preferably at least 80%, more preferably at least 90%, and mostpreferably at least 95% or at least 98% amino acid sequence homologywith a reference translocation domain. The term fragment, when used inrelation to a translocation domain, means a peptide having at least 20,preferably at least 40, more preferably at least 80, and most preferablyat least 100 amino acid residues of the reference translocation domain.In the case of a clostridial translocation domain, the fragmentpreferably has at least 100, preferably at least 150, more preferably atleast 200, and most preferably at least 250 amino acid residues of thereference translocation domain (e.g. H_(N) domain). As with the TM‘fragment’ component (discussed above), translocation ‘fragments’ of thepresent invention embrace fragments of variant translocation domainsbased on the reference sequences.

The Translocation Domain is preferably capable of formation ofion-permeable pores in lipid membranes under conditions of low pH.Preferably it has been found to use only those portions of the proteinmolecule capable of pore-formation within the endosomal membrane.

The Translocation Domain may be obtained from a microbial proteinsource, in particular from a bacterial or viral protein source. Hence,in one embodiment, the Translocation Domain is a translocating domain ofan enzyme, such as a bacterial toxin or viral protein.

It is well documented that certain domains of bacterial toxin moleculesare capable of forming such pores. It is also known that certaintranslocation domains of virally expressed membrane fusion proteins arecapable of forming such pores. Such domains may be employed in thepresent invention.

The Translocation Domain may be of a clostridial origin, such as theH_(N) domain (or a functional component thereof). H_(N) means a portionor fragment of the H-chain of a clostridial neurotoxin approximatelyequivalent to the amino-terminal half of the H-chain, or the domaincorresponding to that fragment in the intact H-chain. In this regard,should it be desired to remove the HC cell-binding function, this may bedone by deletion of the HC or H_(C) amino acid sequence (either at theDNA synthesis level, or at the post-synthesis level by nuclease orprotease treatment). Alternatively, the HC function may be inactivatedby chemical or biological treatment.

Examples of suitable (reference) Translocation Domains include:

Botulinum type A neurotoxin—amino acid residues (449-871)

Botulinum type B neurotoxin—amino acid residues (441-858)

Botulinum type C neurotoxin—amino acid residues (442-866)

Botulinum type D neurotoxin—amino acid residues (446-862)

Botulinum type E neurotoxin—amino acid residues (423-845)

Botulinum type F neurotoxin—amino acid residues (440-864)

Botulinum type G neurotoxin—amino acid residues (442-863)

Tetanus neurotoxin—amino acid residues (458-879)

The above-identified reference sequence should be considered a guide, asslight variations may occur according to sub-serotypes. By way ofexample, US 2007/0166332 (hereby incorporated by reference thereto)cites slightly different clostridial sequences:

Botulinum type A neurotoxin—amino acid residues (A449-K871)

Botulinum type B neurotoxin—amino acid residues (A442-S858)

Botulinum type C neurotoxin—amino acid residues (T450-N866)

Botulinum type D neurotoxin—amino acid residues (D446-N862)

Botulinum type E neurotoxin—amino acid residues (K423-K845)

Botulinum type F neurotoxin—amino acid residues (A440-K864)

Botulinum type G neurotoxin—amino acid residues (S447-S863)

Tetanus neurotoxin—amino acid residues (S458-V879)

Further examples of suitable translocation domains are described indetail in WO 2007/106115, which is hereby incorporated in its entiretyby reference thereto.

In the context of the present invention, a variety of clostridial toxinH_(N) regions comprising a translocation domain can be useful in aspectsof the present invention with the proviso that these active fragmentscan facilitate the release of a non-cytotoxic protease (e.g. aclostridial L-chain) from intracellular vesicles into the cytoplasm ofthe target cell and thus participate in executing the overall cellularmechanism whereby a clostridial toxin proteolytically cleaves asubstrate. The H_(N) regions from the heavy chains of clostridial toxinsare approximately 410-430 amino acids in length and comprise atranslocation domain. Research has shown that the entire length of aH_(N) region from a clostridial toxin heavy chain is not necessary forthe translocating activity of the translocation domain. Thus,embodiments can include clostridial toxin H_(N) regions comprising atranslocation domain having a length of, for example, at least 350 aminoacids, at least 375 amino acids, at least 400 amino acids and at least425 amino acids. Other embodiments can include clostridial toxin H_(N)regions comprising translocation domain having a length of, for example,at most 350 amino acids, at most 375 amino acids, at most 400 aminoacids and at most 425 amino acids.

For further details on the genetic basis of toxin production inClostridium botulinum and C. tetani, we refer to Henderson et al (1997)in The Clostridia: Molecular Biology and Pathogenesis, Academic press.

The term H_(N) embraces naturally-occurring neurotoxin H_(N) portions,and modified H_(N) portions having amino acid sequences that do notoccur in nature and/or synthetic amino acid residues, so long as themodified H_(N) portions still demonstrate the above-mentionedtranslocation function.

Alternatively, the Translocation Domain may be of a non-clostridialorigin. Examples of non-clostridial translocation domain originsinclude, but not be restricted to, the translocation domain ofdiphtheria toxin [O=Keefe et al., Proc. Natl. Acad. Sci. USA (1992) 89,6202-6206; Silverman et al., J. Biol. Chem. (1993) 269, 22524-22532; andLondon, E. (1992) Biochem. Biophys. Acta., 1112, pp. 25-51], thetranslocation domain of Pseudomonas exotoxin type A [Prior et al.Biochemistry (1992) 31, 3555-3559], the translocation domains of anthraxtoxin [Blanke et al. Proc. Natl. Acad. Sci. USA (1996) 93, 8437-8442], avariety of fusogenic or hydrophobic peptides of translocating function[Plank et al. J. Biol. Chem. (1994) 269, 12918-12924; and Wagner et al(1992) PNAS, 89, pp. 7934-7938], and amphiphilic peptides [Murata et al(1992) Biochem., 31, pp. 1986-1992]. The Translocation Domain may mirrorthe Translocation Domain present in a naturally-occurring protein, ormay include amino acid variations so long as the variations do notdestroy the translocating ability of the Translocation Domain.

Particular examples of viral translocation domains suitable for use inthe compositions and methods described herein include certaintranslocating domains of virally expressed membrane fusion proteins. Forexample, Wagner et al. (1992) and Murata et al. (1992) describe thetranslocation (i.e. membrane fusion and vesiculation) function of anumber of fusogenic and amphiphilic peptides derived from the N-terminalregion of influenza virus haemagglutinin. Other virally expressedmembrane fusion proteins known to have the desired translocatingactivity are a translocating domain of a fusogenic peptide of SemlikiForest Virus (SFV), a translocating domain of vesicular stomatitis virus(VSV) glycoprotein G, a translocating domain of SER virus F protein anda translocating domain of Foamy virus envelope glycoprotein. Virallyencoded Aspike proteins have particular application in the context ofthe present invention, for example, the E1 protein of SFV and the Gprotein of the G protein of VSV.

Use of the translocation domains listed in Table 2 (below) includes useof sequence variants thereof. A variant may comprise one or moreconservative nucleic acid substitutions and/or nucleic acid deletions orinsertions, with the proviso that the variant possesses the requisitetranslocating function. A variant may also comprise one or more aminoacid substitutions and/or amino acid deletions or insertions, so long asthe variant possesses the requisite translocating function.

TABLE 2 Translocation Amino acid Domain source residues ReferencesDiphtheria 194-380 Silverman etal., 1994, J. Biol. toxin Chem. 269,22524-22532 London E., 1992, Biochem. Biophys. Acta., 1113, 25-51 DomainII of 405-613 Prior et al., 1992, pseudomonas Biochemistry 31, 3555-3559exotoxin Kihara & Pastan, 1994, Bioconj Chem. 5, 532-538 Influenza virusGLFGAIAGFIENGW Plank et al., 1994, J. Biol. haemagglutinin EGMIDGWYG,and Chem. 269, 12918-12924 Variants thereof Wagner et al., 1992, PNAS,89, 7934-7938 Murata et al., 1992, Biochemistry 31, 1986-1992 SemlikiForest Translocation domain Kielian etal., 1996, J Cell Biol. virusfusogenic 134(4), 863-872 protein Vesicular 118-139 Yao et al., 2003,Virology Stomatitis virus 310(2), 319-332 glycoprotein G SER virus FTranslocation domain Seth et al., 2003, J Virol protein 77(11) 6520-6527Foamy virus Translocation domain Picard-Maureau etal., 2003, J envelopeVirol. 77(8), 4722-4730 glycoprotein

The polypeptides of the compositions and methods described herein mayfurther comprise a translocation facilitating domain. This domainfacilitates delivery of the non-cytotoxic protease into the cytosol ofthe target cell and are described, for example, in WO 08/008803 and WO08/008805, each of which is herein incorporated by reference thereto.

By way of example, suitable translocation facilitating domains includean enveloped virus fusogenic peptide domain, for example, suitablefusogenic peptide domains include influenza virus fusogenic peptidedomain (e.g. influenza A virus fusogenic peptide domain of 23 aminoacids), alphavirus fusogenic peptide domain (e.g. Semliki Forest virusfusogenic peptide domain of 26 amino acids), vesiculovirus fusogenicpeptide domain (e.g. vesicular stomatitis virus fusogenic peptide domainof 21 amino acids), respirovirus fusogenic peptide domain (e.g. Sendaivirus fusogenic peptide domain of 25 amino acids), morbiliivirusfusogenic peptide domain (e.g. Canine distemper virus fusogenic peptidedomain of 25 amino acids), avulavirus fusogenic peptide domain (e.g.Newcastle disease virus fusogenic peptide domain of 25 amino acids),henipavirus fusogenic peptide domain (e.g. Hendra virus fusogenicpeptide domain of 25 amino acids), metapneumovirus fusogenic peptidedomain (e.g. Human metapneumovirus fusogenic peptide domain of 25 aminoacids) or spumavirus fusogenic peptide domain such as simian foamy virusfusogenic peptide domain; or fragments or variants thereof.

By way of further example, a translocation facilitating domain maycomprise a Clostridial toxin H_(N) domain or a fragment or variantthereof. In more detail, a Clostridial toxin H_(N) translocationfacilitating domain may have a length of at least 200 amino acids, atleast 225 amino acids, at least 250 amino acids, at least 275 aminoacids. In this regard, a Clostridial toxin H_(N) translocationfacilitating domain preferably has a length of at most 200 amino acids,at most 225 amino acids, at most 250 amino acids, or at most 275 aminoacids. Specific examples include:

Botulinum type A neurotoxin—amino acid residues (872-1110)

Botulinum type B neurotoxin—amino acid residues (859-1097)

Botulinum type C neurotoxin—amino acid residues (867-1111)

Botulinum type D neurotoxin—amino acid residues (863-1098)

Botulinum type E neurotoxin—amino acid residues (846-1085)

Botulinum type F neurotoxin—amino acid residues (865-1105)

Botulinum type G neurotoxin—amino acid residues (864-1105)

Tetanus neurotoxin—amino acid residues (880-1127)

The above sequence positions may vary somewhat according toserotype/sub-type, and further examples of suitable (reference)Clostridial toxin H_(N) domains include:

Botulinum type A neurotoxin—amino acid residues (874-1110)

Botulinum type B neurotoxin—amino acid residues (861-1097)

Botulinum type C neurotoxin—amino acid residues (869-1111)

Botulinum type D neurotoxin—amino acid residues (865-1098)

Botulinum type E neurotoxin—amino acid residues (848-1085)

Botulinum type F neurotoxin—amino acid residues (867-1105)

Botulinum type G neurotoxin—amino acid residues (866-1105)

Tetanus neurotoxin—amino acid residues (882-1127)

Any of the above-described facilitating domains may be combined with anyof the previously described translocation domain peptides that aresuitable for use in the present invention. Thus, by way of example, anon-clostridial facilitating domain may be combined with non-clostridialtranslocation domain peptide or with clostridial translocation domainpeptide. Alternatively, a Clostridial toxin H_(N) translocationfacilitating domain may be combined with a non-clostridial translocationdomain peptide. Alternatively, a Clostridial toxin H_(N) facilitatingdomain may be combined or with a clostridial translocation domainpeptide, examples of which include:

Botulinum type A neurotoxin—amino acid residues (449-1110)

Botulinum type B neurotoxin—amino acid residues (442-1097)

Botulinum type C neurotoxin—amino acid residues (450-1111)

Botulinum type D neurotoxin—amino acid residues (446-1098)

Botulinum type E neurotoxin—amino acid residues (423-1085)

Botulinum type F neurotoxin—amino acid residues (440-1105)

Botulinum type G neurotoxin—amino acid residues (447-1105)

Tetanus neurotoxin—amino acid residues (458-1127)

The above-identified reference sequences should be considered a guide,as slight variations may occur according to sub-serotypes.

Sequence Homology

Any of a variety of sequence alignment methods can be used to determinepercent identity, including, without limitation, global methods, localmethods and hybrid methods, such as, e.g., segment approach methods.Protocols to determine percent identity are routine procedures withinthe scope of one skilled in the art. Global methods align sequences fromthe beginning to the end of the molecule and determine the bestalignment by adding up scores of individual residue pairs and byimposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W,see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving theSensitivity of Progressive Multiple Sequence Alignment Through SequenceWeighting, Position-Specific Gap Penalties and Weight Matrix Choice,22(22) Nucleic Acids Research 4673-4680 (1994); and iterativerefinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracyof Multiple Protein. Sequence Alignments by Iterative Refinement asAssessed by Reference to Structural Alignments, 264(4) J. Mol. Biol.823-838 (1996). Local methods align sequences by identifying one or moreconserved motifs shared by all of the input sequences. Non-limitingmethods include, e.g., Match-box, see, e.g., Eric Depiereux and ErnestFeytmans, Match-Box: A Fundamentally New Algorithm for the SimultaneousAlignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992);Gibbs sampling, see, e.g., C. E. Lawrence et al., Detecting SubtleSequence Signals: A Gibbs Sampling Strategy for Multiple Alignment,262(5131) Science 208-214 (1993); Align-M, see, e.g., Ivo Van WaIIe etal., Align-M—A New Algorithm for Multiple Alignment of Highly DivergentSequences, 20(9) Bioinformatics:1428-1435 (2004).

Thus, percent sequence identity is determined by conventional methods.See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992.Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “blosum 62” scoring matrix of Henikoff and Henikoff (ibid.) asshown below (amino acids are indicated by the standard one-lettercodes).

Alignment Scores for Determining Sequence Identity

A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2 −2 1 6C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3 −2 −2 6H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2 −3 −4 −1−2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3 −1 0 −2−3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2 −2 −1 −3−1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1 −2 −1 4 T0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4 −2 −2 −3 −2−2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1 −1 −2 −1 3−3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0 −3 −1 4

The percent identity is then calculated as:

$\frac{{Total}\mspace{14mu}{number}\mspace{14mu}{of}\mspace{14mu}{identical}\mspace{14mu}{matches}}{\begin{matrix}\left\lbrack {{length}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{longer}\mspace{14mu}{sequence}\mspace{14mu}{plus}\mspace{14mu}{the}} \right. \\{{number}\mspace{14mu}{of}\mspace{14mu}{gaps}\mspace{14mu}{introduced}\mspace{14mu}{into}\mspace{14mu}{the}\mspace{14mu}{longer}} \\\left. {{sequence}\mspace{14mu}{in}\mspace{14mu}{order}\mspace{14mu}{to}\mspace{14mu}{align}\mspace{14mu}{the}\mspace{14mu}{two}\mspace{14mu}{sequences}} \right\rbrack\end{matrix}} \times 100$

Substantially homologous polypeptides are characterized as having one ormore amino acid substitutions, deletions or additions. These changes arepreferably of a minor nature, that is conservative amino acidsubstitutions (see below) and other substitutions that do notsignificantly affect the folding or activity of the polypeptide; smalldeletions, typically of one to about 30 amino acids; and small amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue, a small linker peptide of up to about 20-25 residues, or anaffinity tag.

Conservative Amino Acid Substitutions

Basic: arginine

-   -   lysine    -   histidine

Acidic: glutamic acid

-   -   aspartic acid

Polar: glutamine

-   -   asparagine

Hydrophobic: leucine

-   -   isoleucine    -   valine

Aromatic: phenylalanine

-   -   tryptophan    -   tyrosine

Small: glycine

-   -   alanine    -   serine    -   threonine    -   methionine

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline and α-methyl serine) may be substituted for amino acidresidues of the polypeptides of the present invention. A limited numberof non-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted forclostridial polypeptide amino acid residues. The polypeptides of thepresent invention can also comprise non-naturally occurring amino acidresidues.

Non-naturally occurring amino acids include, without limitation,trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline,trans-4-hydroxy-proline, N-methylglycine, allo-threonine,methyl-threonine, hydroxy-ethyl cysteine, hydroxyethylhomo-cysteine,nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline,2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and4-fluorophenylalanine. Several methods are known in the art forincorporating non-naturally occurring amino acid residues into proteins.For example, an in vitro system can be employed wherein nonsensemutations are suppressed using chemically aminoacylated suppressortRNAs. Methods for synthesizing amino acids and aminoacylating tRNA areknown in the art. Transcription and translation of plasmids containingnonsense mutations is carried out in a cell free system comprising an E.coli S30 extract and commercially available enzymes and other reagents.Proteins are purified by chromatography. See, for example, Robertson etal., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol.202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al.,Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method,translation is carried out in Xenopus oocytes by microinjection ofmutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti etal., J. Biol. Chem. 271:19991-8, 1996). Within a third method, E. colicells are cultured in the absence of a natural amino acid that is to bereplaced (e.g., phenylalanine) and in the presence of the desirednon-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). Thenon-naturally occurring amino acid is incorporated into the polypeptidein place of its natural counterpart. See, Koide et al., Biochem.33:7470-6, 1994. Naturally occurring amino acid residues can beconverted to non-naturally occurring species by in vitro chemicalmodification. Chemical modification can be combined with site-directedmutagenesis to further expand the range of substitutions (Wynn andRichards, Protein Sci. 2:395-403, 1993).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for amino acid residues ofpolypeptides of the present invention.

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244: 1081-5, 1989). Sites of biological interactioncan also be determined by physical analysis of structure, as determinedby such techniques as nuclear magnetic resonance, crystallography,electron diffraction or photoaffinity labeling, in conjunction withmutation of putative contact site amino acids. See, for example, de Voset al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol.224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. Theidentities of essential amino acids can also be inferred from analysisof homologies with related components (e.g. the translocation orprotease components) of the polypeptides of the present invention.

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenised polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner etal., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Neret al., DNA 7:127, 1988).

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner etal., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Neret al., DNA 7:127, 1988).

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   -   1. A fusion protein comprising: (i) a botulinum neurotoxin (BTx)        or a tetanus neurotoxin (TTx), and (ii) an anthrax toxin        protective antigen (PA), or a C-terminal receptor-binding domain        of PA, wherein part (a) and (b) are linked or fused together.    -   2. The fusion protein of paragraph 1, wherein the BTx or TTx        comprises a BTx or TTx enzymatic moiety and the translocation        signal.    -   3. The fusion protein of paragraph 2, wherein the BTx enzymatic        moiety or translocation signal is selected from the BTx light        chain and heavy chain domains of any one of BTx/A, BTx/B, BTx/C,        BTx/D, BTx/E, BTx/F, BTx/G, and a non-Clostridial botulinum-like        toxin.    -   4. The fusion protein of paragraph 2, wherein the BTx or TTx        enzymatic moiety or translocation signal is selected from the        respective LC enzymatic moiety or H_(N) translocation peptide        found in Table 1.    -   5. The fusion protein of any one of paragraph 2-4, wherein the        part (a) and (b), or the enzymatic moiety and translocation        peptide are linked by a linker peptide.    -   6. The fusion protein of paragraph 5, wherein the linker peptide        is 1-20 amino acids long.    -   7. The fusion protein of paragraph 5 or 6, wherein the linker        peptide is stable in human serum for at least 1 minute.    -   8. The fusion protein of any one of paragraph 5-7, wherein the        linker peptide comprises at least one amino amino acid that is        Gly or Ser.    -   9. The fusion protein of any one of paragraph 5-8, wherein the        linker peptide does not comprise Lys and/or Arg.    -   10. The fusion protein of any one of paragraph 1-9, wherein the        C-terminal receptor-binding domain of PA comprises a PAd4        domain, a PAd2 and a PAd4 domain, or PA63.    -   11. The fusion protein of any one of paragraph 1-10, wherein the        PA or the C-terminal receptor-binding domain of PA is resistant        to cleavage by a protease.    -   12. The fusion protein of any one of paragraph 1-11, wherein the        the PA or the C-terminal receptor-binding domain of PA is        resistant to furin cleavage, or Lys C cleavage or both.    -   13. A fusion protein comprising: (a) a non-cytotoxic protease,        which protease is capable of cleaving a SNARE protein in a        nociceptor neuron; and (b) a protein capable of binding to an        anthrax toxin protective antigen (PA) or a fragment thereof,        wherein the PA or fragment binds a receptor expressed on the        nociceptor neuron.    -   14. The fusion protein of paragraph 13, wherein the        non-cytotoxic protease comprises a clostridial neurotoxin        L-chain.    -   15. The fusion protein of paragraph 14, wherein the clostridial        neurotoxin is botulinum neurotoxin (BTx) or tetanus neurotoxin        (TTx).    -   16. The fusion protein of paragraph 14 or 15, wherein the        clostridial neurotoxin L-chain is selected from Table 1.    -   17. The fusion protein of any one of paragraph 13-16, wherein        the PA-binding receptor expressed on the nociceptor neuron is        ANTXR2 (CMG2).    -   18. The fusion protein of any one of paragraph 13-16, wherein        the protein capable of binding to PA is an anthrax toxin lethal        factor (LF), or an anthrax toxin edema factor (EF).    -   19. The fusion protein of paragraph 18, wherein PA binding        domain of LF is the N-terminal domain of LF, (abbreviated as        LFPABD or LFn).    -   20. The fusion protein of paragraph 18, wherein PA binding        domain of EF is the N-terminal domain of EF, (abbreviated as        EFPABD or EFn).    -   21. A fusion protein comprising: (a) a disulfide-containing        peptide toxin (these include the channel blocking toxins having        a cysteine-knot motif), which is capable of blocking ion        channels in a nociceptor neuron; and (b) a targeting moiety (TM)        that is capable of binding to a binding site on the nociceptor        neuron, wherein the nociceptor neuron expresses said ion        channels therein (e.g., sodium or calcium or both sodium and        calcium);    -   22. The fusion protein of paragraph 21, wherein the        disulfide-containing peptide toxin comprises a cysteine knot        motif.    -   23. The fusion protein of paragraph 21 or 22, wherein the        disulfide-containing peptide toxin is a conotoxin, an agatoxin,        a delta-palutoxin, a huwentotoxin or a ProTx II toxin.    -   24. The fusion protein according to paragraph 21, 22, or 23,        wherein the TM is selected from the group consisting of: an        anthrax toxin protective antigen (PA); a C-terminal        receptor-binding domain of PA; and a nociceptor neuron-binding        protein.    -   25. The fusion protein according to paragraph 24, wherein the PA        or C-terminal receptor-binding domain of PA interacts with and        binds the ANTXR2 (CMG2) receptor expressed on the nociceptor        neuron.    -   26. The fusion protein according to paragraph 24 or 25, wherein        the PA is a mutant or variant PA that is resistant to furin        cleavage.    -   27. The fusion protein according to paragraph 24, 25, or 26,        wherein the C-terminal receptor-binding domain of PA is PA63 or        PAd4 or comprises PAd2 and PAd4 domains.    -   28. The fusion protein according to paragraph 27, wherein the        PAd4 is resistant to cleavage by a protease.    -   29. The fusion protein according to paragraph 28, wherein the        protease is furin or Lys C.    -   30. The fusion protein of any one of paragraph 24-29, wherein        the nociceptor neuron-binding protein is an antibody.    -   31. The fusion protein of paragraph 30, wherein the antibody        specifically binds to nerve growth factor receptor, the ANTXR2        receptor, or an ion-channel protein present on nociceptor        neurons.    -   32. The fusion protein of paragraph 31, wherein the ion-channel        protein is selected from Nav1.7, Nav1.8 or Nav1.9.    -   33. A fusion protein comprising: (a) a disulfide-containing        peptide toxin (this are channel blocking toxin having a        cysteine-knot motif), which is capable of blocking sodium or        calcium or both sodium and calcium channels in a nociceptor        neuron; and (b) a protein capable of binding to an anthrax toxin        protective antigen (PA) or a fragment thereof, wherein the        fragment binds a receptor expressed on the nociceptor neuron.    -   34. The fusion protein of paragraph 33, wherein the        disulfide-containing peptide toxin comprises a cysteine knot        motif.    -   35. The fusion protein of paragraph 33 or 34, wherein the        disulfide-containing peptide toxin is a conotoxin, an agatoxin,        a delta-palutoxin, a huwentotoxin or a ProTx II toxin.    -   36. The fusion protein of paragraph 33, 34 or 35, wherein the        PA-binding receptor expressed on the nociceptor neuron is ANTXR2        (CMG2).    -   37. The fusion protein of any one of paragraph 33-37, wherein        the protein capable of binding to PA is: an anthrax toxin lethal        factor (LF); or an anthrax toxin edema factor (EF).    -   38. A fusion protein comprising: (a) an AB toxin; (b) an anthrax        toxin protective antigen (PA) or a fragment thereof, wherein the        PA or fragment binds a receptor expressed on the nociceptor        neuron; and (c) a translocation domain (TL) that is capable of        translocating the protease from within an endosome, across the        endosomal membrane and into the cytosol of the nociceptor        neuron.    -   39. The fusion protein of paragraph 38, wherein the AB toxin is        selected from Ricin toxin, Cholera toxin A-part and B-part;        Pseudomonas aeruginosa Exotoxin A A-part and B-part; Shiga toxin        A-part and B-part; and Diphtheria toxin A-part and B-part.    -   40. The fusion protein of paragraph 38 or 39, wherein the        PA-binding receptor expressed on the nociceptor neuron is ANTXR2        (CMG2).    -   41. The fusion protein of paragraph 38, 30, or 40, wherein the        PA fragment is a C-terminal receptor-binding domain of PA.    -   42. The fusion protein of any one of paragraph 38-41, wherein        the TL is a clostridial neurotoxin translocation domain; a        holotoxin; or a variant form of the holotoxin that has the toxin        receptor-binding function of the AB toxin negated.    -   43. A nucleic acid encoding a fusion protein according to any of        the previous paragraphs.    -   44. A vector comprising the nucleic acid of paragraph 43.    -   45. The vector of paragraph 44, wherein the vector is a plasmid,        a bacteriophage, a phagmid, a cosmid, a viral vector, or a viral        particle.    -   46. A cell comprising the nucleic acid of paragraph 43 or the        vector of paragraph 44 or 45.    -   47. A method of producing the fusion protein of any of the        preceding claim comprising: culturing the cell of paragraph 46        in conditions such that the fusion protein is expressed; and        recovering the fusion protein.    -   48. The fusion protein produced by the method of paragraph 47.    -   49. A composition comprising the fusion protein of any one of        previous paragraphs.    -   50. A method for treatment of pain, the method comprising        administering to a subject in need thereof the composition of        any of the preceding paragraphs.    -   51. A method of treating pain comprising administering to a        subject in need thereof, native mature anthrax toxin protective        antigen (PA) and anthrax toxin edema factor (EF), anthrax toxin        lethal factor (LF) or any combination thereof.    -   52. A fusion protein comprising a botulinum neurotoxin (BTx)        moiety comprising an N-terminal enzymatic domain (L chain) and        an intermediate pore-forming/translocation-domain (H_(N)) of the        BTx linked to a C-terminal receptor-binding domain of anthrax        toxin protective antigen (PAd4 domain).    -   53. The fusion protein of paragraph 52 further comprising a        linker peptide between the BTx moiety and the PAd4 domain.    -   54. The fusion protein of paragraph 53, wherein the linker        peptide is 1-20 amino acids long.    -   55. The fusion protein of paragraph 53 or 54, wherein the linker        peptide is stable in human serum for at least 1 minute.    -   56. The fusion protein of any one of paragraph 53-55, wherein        the linker peptide comprises at least one amino amino acid that        is Gly or Ser.    -   57. The fusion protein of any one of paragraph 53-56, wherein        the linker peptide does not comprise Lys and/or Arg.    -   58. The fusion protein of any one of paragraph 52-57, wherein        the BTx moiety is selected from the BTx light chain and heavy        chain domains of any one of BTx/A, BTx/B, BTx/C, BTx/D, BTx/E,        BTx/F, BTx/G, and first non-Clostridial botulinum-like toxin.    -   59. The fusion protein of any one of paragraph 52-58, wherein        the fusion protein comprising 2-10 PAd4 domains in tandem.    -   60. The fusion protein of any one of paragraph 52-59, wherein        1-60 consecutive amino acids from the N-terminal side adjacent        to the native PAd4 domain are further incorporated between the        BTx moiety and the PAd4.    -   61. The fusion protein of any one of paragraph 52-60, wherein        the one or more of the Lys residues in the PAd4 domain at        positions 594, 613, 633, 637, 653, 673, 679, 680, 684, 695, 703,        722, 723, and 729, and 730 of SEQ ID NO:1 (minus the 29 aa        signal peptide in SEQ. ID. NO:1) have been replaced by Arg or        His.    -   62. The fusion protein of any one of paragraph 52-61, wherein        one or more, up to and including each of the Lys residues in the        PAd4 domain at positions 623, 642, 662, 666, 682, 702, 708, 709,        713, 724, 732, 751, 752, 758, and 759 in SEQ. ID. NO:1 have been        replaced Arg or His.    -   63. The fusion protein of any one of paragraph 52-63 comprising        an entire PA protein, wherein the furin cleavage site comprising        amino acid residues RKKR has been replaced by a furin-resistant        amino acid sequence, wherein RKKR are the residues 164-167 of        SEQ ID NO: 1 minus the 29 amino acid signal peptide in SEQ ID        NO:1.    -   64. The fusion protein of paragraph 63, wherein the        furin-resistant amino acid sequence is SSSR (SEQ ID NO: 32) or        SSSS (SEQ ID NO: 33).    -   65. The fusion protein of any one of paragraph 52-64, wherein        one or more of the Asn residues in the PAd4 domain at position        601, 713, 719 of SEQ ID NO:1 (the numbering is that after minus        the 29 aa signal peptide from SEQ. ID. NO:1) have been replaced        by Asp.    -   66. The fusion protein of any one of paragraph 52-65, further        comprising at least one D-amino acid at the N-terminus of the        fusion protein.    -   67. The fusion protein of any one of paragraph 52-66, wherein        the residue corresponding to the junction between the L chain of        BTx with the H_(N) chain of BTx has been cleaved.    -   68. A composition comprising the fusion protein of any one of        paragraphs 52-67.    -   69. The composition of paragraph 68 further comprising a        pharmaceutically acceptable carrier, excipient or diluent.    -   70. A fusion protein comprising: a botulinum neurotoxin        N-terminal enzymatic domain of a botulinum neurotoxin (BTx)        moiety (L chain of BTx), and a N-terminal domain (LFn) of        anthrax toxin lethal factor, which domain binds to oligomeric        forms of PA63, the proteolytically activated form of anthrax PA;        or the N-terminal domain (EFn) of anthrax toxin edema factor,        which domain binds to oligomeric forms of PA63, the        proteolytically activated form of anthrax PAthe enzymatic        domain, wherein part (a) is linked N-terminally or C-terminally        or both N-terminally and C-terminally to part (b).    -   71. The fusion protein of paragraph 70 further comprising an        amino acid sequence defining a belt corresponding to the        N-terminal part of the BTx H_(N) domain which is located at        C-terminal side of the BTx moiety, and the belt is linked to the        L chain.    -   72. The fusion protein of paragraph 71, wherein the residue        corresponding to the L chain junction with the belt of the BTx        H_(N) domain has been cleaved.    -   73. The fusion protein of paragraph 71 or 72, wherein the Cys        residues in the BTx L chain and the belt corresponding to the        N-terminal part of the BTx H_(N) domain have been changed to        Ala, Ser, or Thr.    -   74. The fusion protein of any one of paragraph 70-73, further        comprising at least one D-amino acid at the N-terminus of the        fusion protein.    -   75. The fusion protein of any one of paragraph 70-73, further        comprising a linker peptide between the BTx L moiety and the LFn        or EFn domain.    -   76. The fusion protein of paragraph 75, wherein the linker        peptide is 1-20 amino acids long.    -   77. The fusion protein of paragraph 75 or 76, wherein the linker        peptide is stable in human serum for at least 1 minute.    -   78. The fusion protein of any one of paragraph 75-78, wherein        the linker peptide comprises at least one amino amino acid that        is Gly or Ser.    -   79. The fusion protein of any one of paragraph 75-79, wherein        the linker peptide does not comprise Lys or Arg.    -   80. A composition comprising any one of the fusion proteins of        paragraphs 70-79.    -   81. The composition of claim 80 further comprising a        pharmaceutically acceptable carrier, excipient or diluent.    -   82. The composition of paragraph 80 or 81 and a native anthrax        toxin protective antigen (PA) protein.    -   83. The composition of paragraph 82, wherein the PA protein is        an oligomeric PA.    -   84. The composition of paragraph 83, wherein the oligomeric PA        is bound to the fusion protein.    -   85. The composition of any one of paragraphs 80-84 further        comprising a pharmaceutically acceptable carrier excipient or        diluent.    -   86. A fusion protein comprising: anthrax toxin protective        antigen (PA); an anthrax toxin protective antigen C-terminal        receptor binding domain (PAd4); or a nociceptor neuron-binding        protein linked to, a disulfide-containing peptide toxin.    -   87. The fusion protein of paragraph 86, wherein the        disulfide-containing peptide toxin is an inhibitor cysteine knot        toxin.    -   88. The fusion protein of paragraph 86 or 87, wherein the        disulfide-containing peptide toxin is a conotoxin, an agatoxin,        a delta-palutoxin, a huwentotoxin or a ProTx II toxin.    -   89. The fusion protein of paragraph 86, 87 or 88, further        comprising a linker peptide between the PA, PAd4 or        nociceptor-binding protein and the inhibitor cysteine knot        toxin.    -   90. The fusion protein of paragraph 89, wherein the linker        peptide is stable in human serum for at least 1 minute.    -   91. The fusion protein of paragraph 89 or 90, wherein the linker        peptide comprises at least one amino acid that is Gly or Ser.    -   92. The fusion protein of any of the preceding paragraph 89-91,        wherein the linker peptide does not comprise Lys and/or Arg.    -   93. The fusion protein of any of the preceding paragraph 89-92,        wherein the fusion protein comprises 2-10 PAd4 domains in        tandem.    -   94. The fusion protein of any of the preceding paragraph 89-93,        wherein the one or more of the Lys residues in PAd4 or PA at        positions 594, 613, 633, 637, 653, 673, 679, 680, 684, 695, 703,        722, 723, 729, and 730 of SEQ ID NO:1 (minus the 29 aa signal        peptide in SEQ. ID. NO:1) have been replaced by Arg or His.    -   95. The fusion protein of paragraph 94, wherein one or more, up        to and including each of the Lys residues in the PAd4 domain at        positions 623, 642, 662, 666, 682, 702, 708, 709, 713, 724, 732,        751, 752, 758, and 759 in SEQ. ID. NO:1 have been replaced Arg        or His.    -   96. The fusion protein of any of the preceding paragraph 89-95        comprising an entire PA protein, wherein the furin cleavage site        comprising amino acid residues ¹⁶⁴RKKR¹⁶⁷ of SEQ ID NO: 1 has        been replaced by a furin-resistant amino acid sequence, wherein        RKKR are the residues 164-167 of SEQ ID NO: 1 minus the 29 amino        acid signal peptide in SEQ ID NO:1.    -   97. The fusion protein of paragraph 96, wherein the        furin-resistant amino acid sequence is SSSR or SSSS.    -   98. The fusion protein of any of the preceding paragraph 89-97,        wherein one or more of the Asn residues in PAd4 or PA at        position 601, 713, 719 of SEQ ID NO:1 (minus the 29 aa signal        peptide in SEQ. ID. NO:1) have been replaced by Asp.    -   99. The fusion protein of any of the preceding paragraph 89-98,        further comprising at least one D-amino acid at the N-terminus        of the fusion protein.    -   100. The fusion protein of any of the preceding paragraph 89-99,        wherein the nociceptor neuron-binding protein is an antibody.    -   101. The fusion protein of paragraph 100, wherein the antibody        specifically binds to nerve growth factor receptor or an        ion-channel protein present on nociceptor neurons.    -   102. The fusion protein of paragraph 101, wherein the        ion-channel protein is selected from Nav1.7, Nav1.8 or Nav1.9.    -   103. A composition comprising the fusion protein of any one of        the paragraphs 86-102.    -   104. The composition of claim 103 further comprising a        pharmaceutically acceptable carrier, diluent or excipient.    -   105. A fusion protein comprising a disulfide-containing peptide        toxin operably linked N-terminally or C-terminally or both        N-terminally and C-terminally, or chemically crosslinked at one        or more sites to the N-terminal domain (LFn) of anthrax toxin        lethal factor, which domain binds to oligomeric forms of PA63,        the proteolytically activated form of anthrax PA; or the        N-terminal domain (EFn) of anthrax toxin edema factor, which        domain binds to oligomeric forms of PA63, the proteolytically        activated form of anthrax PA.    -   106. The fusion protein of paragraph 105, further comprising a        linker peptide between the LFn and the toxin or the EFn and the        toxin.    -   107. The fusion protein of paragraph 106, wherein the linker        peptide is stable in human serum for at least 1 minute.    -   108. The fusion protein of paragraph 106 or 107, wherein the        linker peptide comprises at least one amino acid that is Gly or        Ser.    -   109. The fusion protein of paragraph 106, 107, or 108, wherein        the linker peptide does not comprise Lys and/or Arg.    -   110. A composition comprising the fusion protein of any one of        claims paragraphs 105-109.    -   111. The composition of paragraph 110 further comprising a        pharmaceutically acceptable carrier excipient or diluent.    -   112. The composition of paragraph 110 or 111 further comprising        a native anthrax toxin protective antigen (PA).    -   113. The composition of paragraph 112, wherein the PA is in an        oligomeric PA.    -   114. The composition of paragraph 113, wherein the oligomeric PA        is bound to the fusion protein.    -   115. The composition of any one of paragraphs 110-114 further        comprising a pharmaceutically acceptable carrier excipient or        diluent.    -   116. A fusion protein comprising an AB toxin fused to a linker        peptide operably linked to a C-terminal receptor-binding domain        of anthrax toxin protective antigen (PAd4 domain), wherein the        fusion protein further comprises a translocation domain, a        holotoxin or a mutant form of the holotoxin that has the toxin        receptor-binding function of the AB toxin negated.    -   117. The fusion protein of paragraph 116, wherein the AB toxin        is selected from Ricin toxin, Cholera toxin A-part and B-part;        Pseudomonas aeruginosa Exotoxin A A-part and B-part; Shiga toxin        A-part and B-part; and Diphtheria toxin A-part and B-part.    -   118. The fusion protein of paragraph 116 or 117, wherein the        linker peptide is 1-20 amino acids long.    -   119. The fusion protein of any of the preceding paragraph        116-118, wherein the linker peptide is stable in human serum for        at least 1 minute.    -   120. The fusion protein of any one of paragraphs 116-119,        wherein the linker peptide comprises at least one amino acid        that is Gly or Ser.    -   121. The fusion protein of any one of paragraphs 116-120,        wherein the linker peptide does not comprise Lys and Arg.    -   122. The fusion protein of any of the preceding paragraph        116-121, wherein 1-60 consecutive amino acids from the        N-terminal side adjacent to the native PAd4 domain are further        incorporated between the AB toxin and the PAd4.    -   123. The fusion protein of any of the preceding paragraph        116-122, wherein one or more of the Asn residues in PAd4 domain        at position 601, 713, 719 of SEQ ID NO: 1 (minus the 29 aa        signal peptide in SEQ. ID. NO:1) have been replaced by Asp.    -   124. The fusion protein of any one of paragraphs 116-123,        further comprising at least one D-amino acid at the N-terminus        of the fusion protein.    -   125. The fusion protein of any one of paragraphs 116-124        comprising 2-10 PAd4 domains in tandem.    -   126. The fusion protein of any of the preceding paragraph        116-125, wherein the one or more of the Lys residues in PAd4        domain at positions 594, 613, 633, 637, 653, 673, 679, 680, 684,        695, 701, 713, 719, 722, 723, 729, and 730 of SEQ ID NO:1 (minus        the 29 aa signal peptide in SEQ. ID. NO:1) (PAd4) have been        replaced by Arg or His.    -   127. The fusion protein of any of the preceding paragraph        116-126, wherein one or more, up to and including each of the        Lys residues in the PAd4 domain at positions 623, 642, 662, 666,        682, 702, 708, 709, 713, 724, 732, 751, 752, 758, and 759 in        SEQ. ID. NO:1 have been replaced Arg or His.    -   128. The fusion protein of any of the preceding paragraph        116-127 comprising an entire PA protein, wherein the furin        cleavage site comprising amino acid residues RKKR has been        replaced by a furin-resistant amino acid sequence, wherein RKKR        are the residues 164-167 of SEQ ID NO: 1 minus the 29 amino acid        signal peptide in SEQ ID NO:1.    -   129. The fusion protein of paragraph 128, wherein the        furin-resistant amino acid sequence is SSSR or SSSS.    -   130. A composition comprising the fusion protein of any one of        paragraphs 114-129.    -   131. The composition of paragraph 130 further comprising a        pharmaceutically acceptable carrier, excipient or diluent.    -   132. A fusion protein comprising an N-terminal enzymatic domain        (Chain A) together with the translocation/pore-forming domain        from a tetanus neurotoxin (TTx) operably linked to a C-terminal        receptor-binding domain of anthrax toxin protective antigen        (PAd4 domain).    -   133. The fusion protein of paragraph 132 further comprising a        linker peptide between the TTx moiety and the PAd4 domain.    -   134. The fusion protein of paragraph 133, wherein the linker        peptide is 1-20 amino acids long.    -   135. The fusion protein of paragraph 133 or134, wherein the        linker peptide is stable in human serum for at least 1 minute.    -   136. The fusion protein of paragraph 133, 134, or 135, wherein        the linker peptide comprises at least one amino amino acid that        is Gly or Ser.    -   137. The fusion protein of any of the preceding paragraph        133-136, wherein the linker peptide does not comprise Lys or        Arg.    -   138. The fusion protein of any one of paragraphs 132-137        comprising two to ten PAd4 domains in tandem.    -   139. The fusion protein of any one of paragraphs 132-138,        wherein 1-60 amino consecutive amino acids from the N-terminal        side adjacent to the native PAd4 domain are further incorporated        between the BTx moiety and the PAd4.    -   140. The fusion protein of any one of paragraphs 132-139,        wherein the one or more of the Lys residues in the PAd4 domain        at positions 594, 613, 633, 637, 653, 673, 679, 680, 684, 695,        701, 713, 719, 722, 723, 729, and 730 of SEQ ID NO:1 (minus the        29 aa signal peptide in SEQ. ID. NO:1) have been replaced by Arg        or His.    -   141. The fusion protein of any one of paragraphs 132-140,        wherein one or more, up to and including each of the Lys        residues in the PAd4 domain at positions 623, 642, 662, 666,        682, 702, 708, 709, 713, 724, 732, 751, 752, 758, and 759 in        SEQ. ID. NO:1 have been replaced Arg or His.    -   142. The fusion protein of any one of paragraphs 132-141,        comprising an entire PA protein, wherein the furin cleavage site        comprising amino acid residues RKKR has been replaced by a        furin-resistant amino acid sequence, wherein RKKR are the        residues 164-167 of SEQ ID NO: 1 minus the 29 amino acid signal        peptide in SEQ ID NO:1.    -   143. The fusion protein of paragraph 142, wherein the        furin-resistant amino acid sequence is SSSR or SSSS.    -   144. The fusion protein of any one of paragraphs 132-143,        wherein one or more of the Asn residues in the PAd4 domain at        position 601, 713, 719 of SEQ ID NO:1 (minus the 29 aa signal        peptide in SEQ. ID. NO:1) have been replaced by Asp.    -   145. The fusion protein of any one of paragraphs 132-144,        further comprising at least one D-amino acid at the N-terminus        of the fusion protein.    -   146. The fusion protein of any one of paragraphs 132-145,        wherein the residue corresponding to a light chain junction of        TTx with a heavy chain of TTx has been cleaved.    -   147. A composition comprising the fusion protein of any one of        paragraphs 132-146.    -   148. The composition of paragraph 147 further comprising a        pharmaceutically acceptable carrier, excipient or diluent.    -   149. A nucleic acid encoding any of the fusion protein of any of        the paragraphs 52-67, 70-79, 86-102, 105-109, 116-129, and        132-146.    -   150. A vector comprising the nucleic acid of paragraph 149.    -   151. The vector of paragraph 150, wherein the vector is a        plasmid, a bacteriophage, a phagmid, a cosmid, a viral particle,        or viral vector.    -   152. A cell comprising the nucleic acid of paragraph 149 or the        vector of paragraph 150 or 151.    -   153. A method of producing the fusion protein of any of the        preceding claim comprising: (i) culturing the cell of paragraph        152 in conditions such that the fusion protein is expressed;        and (ii) recovering the fusion protein.    -   154. The fusion protein produced by the method of paragraph 153.    -   155. The fusion protein of any of the paragraphs 1-42, 52-67,        70-79, 86-102, 105-109, 116-129, and 132-146, which is        glycosylated.    -   156. The fusion protein of any of the paragraphs 1-42, 52-67,        70-79, 86-102, 105-109, 116-129, and 132-146, which is        non-glycosylated.    -   157. The method of paragraph 153, wherein the cell is a        bacterial cell.    -   158. The method of paragraph 157, wherein the bacterial cell is        an Escherichia coli.    -   159. The method of paragraph 153, wherein the cell is a yeast        cell.    -   160. The method of paragraph 159, wherein the yeast is        Saccharomyces cerevisiae.    -   161. The method of paragraph 159, wherein the yeast is cell        glycosylation deficient.    -   162. The method of paragraph 159, wherein the yeast is        glycosylation and protease deficient.    -   163. The method of paragraph 153, wherein said cell is a        mammalian cell.    -   164. The method of paragraph 163, wherein said mammalian cell is        a COS cell, a CHO cell, or a NSO cell.    -   165. A use of any of the fusion proteins of the preceding        paragraphs for the treatment of pain.    -   166. A use of any of the fusion proteins of the preceding        paragraphs for the manufacture of a medicament for the treatment        of pain.    -   167. A method for treatment of pain, the method comprising        administering to a subject in need thereof the composition        selected from paragraphs 49, 68-69, 80-85, 103-104, 110-115,        130-131 and 147-148.    -   168. The method of paragraph 167, wherein the administering is        performed by intrathecal infusion or intra-cerebroventricular        infusion or by an epidural injection into the central nervous        system, or by peripheral administration using intradermal        injection, subcutaneous injection, intramuscular injection,        intraneural injection, or intra-articular injection.    -   169. The method of paragraph 168, wherein the pain is selected        from diabetic neuropathic pain, cancer pain, fibromyalgia and        other systemic pain disorders.    -   170. A method for treatment of nerve, joint, skin, visceral,        bladder, or muscle pain comprising administering peripherally by        intradermal injection, subcutaneous injection, intramuscular        injection, intraneural injection, or intra-articular injection        to a subject in need thereof the composition selected from        paragraphs 49, 68-69, 80-85, 103-104, 110-115, 130-131 and        147-148.    -   171. A method for treatment of diabetic neuropathic pain, cancer        pain, fibromyalgia or other systemic pain disorders comprising        administering by epidural injection, intrathecal infusion or        intra-cerebroventricular infusion into the central nervous        system of a subject in need thereof the composition selected        from paragraphs 49, 68-69, 80-85, 103-104, 110-115, 130-131 and        147-148.    -   172. The method of any one of paragraphs 167-171, wherein the        composition of claims 80-85 and 110-115 is administered        separately before, simultaneously or after administering a        composition comprising an anthrax protective antigen (PA) in a        pharmaceutically acceptable carrier, excipient or diluent.    -   173. A method of treating pain comprising administering to a        subject in need thereof, native mature anthrax toxin protective        antigen (PA) and anthrax toxin edema factor (EF), anthrax toxin        lethal factor (LF) or any combination thereof.    -   174. A method of manufacture of a pharmaceutical composition for        the treatment of pain, wherein the pharmaceutical composition        comprising one or more of the fusion proteins described in the        preceding paragraphs and a pharmaceutically acceptable carrier        or excipient.    -   175. An engineered fusion protein comprising an anthrax toxin        Protective-Antigen (PA) moiety or its receptor binding domain        (Pad4) fused with an inhibitor cysteine knot (ICK) toxin (e.g.,        a Conotoxin (CTx)).    -   176. An engineered fusion protein comprising an anthrax toxin        lethal factor domain (LFn) fused with an inhibitor cysteine knot        (ICK) toxin (e.g., a Conotoxin (CTx)) and a Protective-Antigen        (PA) moiety.    -   177. An engineered fusion protein comprising an anthrax        protective antigen (PA) moiety or a mutant anthrax protective        antigen (mPA) moiety that has been altered to block its native        receptor-binding function fused with a molecule capable of        specifically targeting a nociceptor surface receptor or an ion        channel receptor and an anthrax lethal factor domain (LFn) fused        to an intracellularly acting toxin catalytic domain.    -   178. An engineered fusion protein comprising an anthrax        protective antigen (PA) moiety or at anthrax protective antigen        (mPA) moiety fused with a molecule capable of specifically        targeting a nociceptor surface receptor or an ion channel        receptor and an anthrax lethal factor domain (LFn) fused to an        intracellularly acting toxin catalytic domain.    -   179. The engineered fusion protein of paragraph 177 or 178,        wherein the molecule is selected from an antibody that        specifically binds to the nerve growth factor receptor, or an        antibody that specifically binds to Nav1.7, Nav1.8 or Nav1.9.    -   180. The engineered fusion protein of any of the preceding        paragraphs, wherein the intracellularly acting toxin catalytic        domain is selected from diphtheria toxin (DTx), Pseudomonas        aeruginosa exotoxin A (PTx), botulinum toxin (BTx) tetanus toxin        (TTx), shiga toxin, ricin toxin, lethal toxin (lethal factor),        and/or Edema toxin (edema factor).    -   181. An engineered fusion protein comprising a native protective        antigen (PA) or a modified (e.g., chemically) or mutant PA        (mPA), wherein the mPA native receptor-binding function is        blocked, and a molecule that can target nociceptor neuron        surface molecules specifically in combination with anthrax toxin        edema factor (EF) and/or lethal factor (LF).    -   182. The engineered fusion protein of any of the preceding        paragraphs, wherein PA or mPA is in an oligomeric form.    -   183. The engineered fusion protein of paragraph 182, wherein the        oligomeric form is bound to the molecule.    -   184. A composition comprising an engineered fusion protein        comprising an anthrax toxin Protective-Antigen (PA) moiety or        its receptor binding domain (Pad4) fused with an inhibitor        cysteine knot (ICK) toxin (e.g., a Conotoxin (CTx)).    -   185. A composition comprising an engineered fusion protein        comprising an anthrax toxin lethal factor domain (LFn) fused        with an inhibitor cysteine knot (ICK) toxin (e.g., a Conotoxin        (CTx)) and a Protective-Antigen (PA) moiety.    -   186. A composition comprising engineered fusion protein        comprising a native protective antigen (PA) or a mutant PA        (mPA), fused, with a molecule capable of specifically targeting        a nociceptor surface receptor or an ion channel receptor and an        anthrax lethal factor domain (LFn) fused to an intracellularly        acting toxin catalytic domain, wherein the mPA has been modified        (e.g., chemically) or mutated so that the PA native        receptor-binding function is blocked.    -   187. The composition of paragraph 186, wherein the molecule is        selected from a nerve growth factor, and an antibody that        specifically binds to Nav1.7, Nav1.8 or Nav1.9.    -   188. The composition of paragraph 186 or 187, wherein the        intracellularly acting toxin catalytic domain is selected from        diphtheria toxin (DTx), Pseudomonas aeruginosa exotoxin A (PTx),        botulinum toxin (BTx) tetanus toxin (TTx), Shiga toxin, ricin        toxin, lethal toxin (lethal factor), and/or Edema toxin (edema        factor).    -   189. A composition comprising an engineered fusion protein        comprising a native protective antigen (PA) or a mutant PA        (mPA), and a molecule that can target nociceptor neuron surface        molecules specifically in combination with anthrax toxin edema        factor (EF), wherein the mPA has been modified (e.g.,        chemically) or mutated so that the PA native receptor-binding        function is blocked.    -   190. The composition of any of the preceding paragraphs, wherein        PA or mPA is in an oligomeric form.    -   191. The composition of paragraph 190, wherein the oligomeric        form is bound to the molecule.    -   192. The composition of any of the preceding paragraphs, the        composition further comprising a pharmaceutically acceptable        carrier or excipient.    -   193. A method for treatment of pain, the method comprising        administering to a subject in need thereof an effective, pain        reducing amount of a composition comprising an engineered fusion        protein comprising an anthrax toxin Protective-Antigen (PA)        moiety or its receptor binding domain (PAd4) fused to an        intracellularly acting toxin catalytic domain, wherein the        engineered fusion protein is delivered to nociceptor neurons and        results in decreased intracellular signaling events in the        nociceptor neurons or decreased neurotransmitter release from        the nociceptor neurons.    -   194. The method of paragraph 193, wherein the intracellularly        acting toxin catalytic domain is selected from diphtheria toxin        (DTx), Pseudomonas aeruginosa exotoxin A (PTx), botulinum toxin        (BTx) tetanus toxin (TTx), Shiga toxin, ricin toxin, lethal        toxin (lethal factor), and/or Edema toxin (edema factor).    -   195. A method for treatment of pain, the method comprising        administering to a subject in need thereof an effective, pain        reducing amount of a composition comprising an engineered fusion        protein comprising an anthrax toxin Protective-Antigen (PA)        moiety or its receptor binding domain (PAd4) fused with an        inhibitor cysteine knot (ICK) toxin (e.g., a Conotoxin (CTx)).    -   196. A method for treatment of pain, the method comprising        administering to a subject in need thereof an effective amount        of a composition comprising an engineered fusion protein        comprising an anthrax toxin lethal factor (LFn) fused with an        inhibitor cysteine knot (ICK) toxin (e.g., a Conotoxin (CTx))        and a Protective-Antigen (PA) moiety.    -   197. The composition of any of the preceding paragraphs, the        composition further comprising a pharmaceutically acceptable        carrier or excipient.    -   198. A method for treatment of pain, the method comprising        administering to a subject in need thereof an effective, pain        reducing amount of an engineered fusion protein comprising an        anthrax protective antigen (PA) moiety or a mutant anthrax        protective antigen (mPA) moiety that has been altered to block        its native receptor-binding function fused with a molecule        capable of specifically targeting a nociceptor surface receptor        or an ion channel receptor and an anthrax lethal factor domain        (LFn) fused to an intracellularly acting toxin catalytic domain.    -   199. The method of paragraph 198, wherein the molecule is        selected from an antibody that specifically binds to the nerve        growth factor receptor and an antibody that specifically binds        to Nav1.7, Nav1.8 or Nav1.9.    -   200. The method of paragraph 198 or 199, wherein the        intracellularly acting toxin catalytic domain is selected from        diphtheria toxin (DTx), Pseudomonas aeruginosa exotoxin A (PTx),        botulinum toxin (BTx) tetanus toxin (TTx), Shiga toxin, ricin        toxin, lethal toxin (lethal factor), and/or Edema toxin (edema        factor).    -   201. A method of treating pain in a subject in need thereof        comprising administering to the subject an engineered fusion        protein comprising a native protective antigen (PA) or a mutant        PA (mPA), and a molecule that can target nociceptor surface        molecules specifically in combination with anthrax toxin edema        factor (EF) and/or lethal factor (LF), wherein the mPA has been        modified (e.g., chemically) or mutated so that the PA native        receptor-binding function is blocked.    -   202. The method of any of the preceding paragraphs, wherein the        PA or mPA is administered in an oligomeric form, wherein the        oligomeric PA or mPA is formed from proteolytically activated PA        or mPA (or mutant thereof) to achieve increased avidity for        receptor-bearing cells.    -   203. The method of paragraph 202, wherein the oligomeric form is        bound to the molecule before administering.    -   204. The method of paragraph 202 or 203, wherein the oligomeric        form is administered in a separate injection before,        simultaneously or after administering the “effector molecule.”

The technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.

EXAMPLES Example 1

We have discovered that the major anthrax toxin receptor, ANTXR2, ishighly and specifically expressed by nociceptor neurons compared toother neuronal subtypes and CNS tissues. By using the endosomal deliverymechanisms inherent to anthrax toxin, we can specifically delivermolecular cargo into nociceptors that would result in pain-specificblock without causing other neurological side effects.

Based on this discovery, we provide: (1) pain-specific blockade andtargeted analgesic mechanisms in chronic pain conditions. Theseconditions include osteoarthritis, spasticity, rheumatoid arthritis,chemotherapy induced neuropathy, and cancer pain. (2) Treatment ofmuscle spasticity. Pain is a major component of spastic diseaseconditions. Dysregulation of sensory-motor reflex circuits triggered bynociceptors could drive muscle spasticity. We hypothesize that anthraxtoxin mediated nociceptor silencing may not only treat pain but alsodecrease muscle spasticity. (3) Treatment of osteoarthritic conditions.Joint pain and destruction is a major component of osteoarticulardiseases and rheumatological diseases. Outside the nervous system (whereit is specific to nociceptors), we find that ANTXR2 is highly expressedby macrophages, osteoblasts, and osteoclasts, which are keyhematopoietic cells mediating joint. The anthrax toxin and theirdelivery mechanisms can used to specifically target joint pain throughnociceptors without neurological side effects, as well as concurrentlytargeting the macrophages, osteoblasts and osteoclasts that mediatejoint destruction.

To determine if anthrax toxin can be used to specifically targetselected molecular cargo proteins into nociceptor neurons through itsbuilt in endosomal escape and cytosolic delivery mechanisms. We willfurther test if anthrax toxin can be used to silence chronic pain inseveral disease conditions (osteoarthritis, muscle injury, rheumatoidarthritis, chemotherapy induced neuropathy, cancer pain), and as well asjoint destruction in osteoarthritis.

We recently discovered that ANTXR2 is highly expressed in nociceptorneurons, and is specific to these neurons compared to other neuronalsubtypes, based on our detailed FACS purified somatosensory neuronexpression databases, in situ hybridization databases, and tissueexpression databases. ANTXR2 is also highly expressed in macrophages,osteoblasts, and osteoclasts, key cell-types in joint destruction. Thisexpression potentially allows anthrax toxin to deliver cargo into thesecells to slow joint destruction in osteoarthritis.

We are utilizing anthrax toxin or its delivery mechanisms tospecifically block chronic pain or joint destruction in differentdisease conditions. In some applications we will use native anthraxtoxin, consisting of protective antigen (PA), the receptor bindingcomponent, together with Lethal Factor (LF, which silences MAP kinases)or Edema Factor (EF, a calcium dependent adenylate cyclase). In otherapplications, we will use anthrax toxin as a platform to mediatecytoplasmic delivery of the enzymatic moieties of other bacterialtoxins, including diphtheria toxin (DTA) or ricin (Rcn).

Native anthrax toxin (PA, LF, EF) to silence pain. Anthrax skininfections cause lesions that are painless. PA binding to ANTXR2mediates the delivery of Lethal Factor (LF), which is known to block MAPkinases or edema factor (EF), which is an adenylate cyclase. Both MAPkinase and cAMP pathways mediate nociceptor sensitization and chronicpain, and their modulation could silence pain. Thus, we will utilizelocal subcutaneous or joint injections of combinations of PA+LF, PA+EF,or PA+LF+EF to induce pain blockade in different disease conditions.

Anthrax toxin mediated cytosolic delivery of enzymatic moieties ofdiphtheria toxin (DTA) or Ricin (Rcn) to silence pain and arthriticjoint destruction: PA, the receptor binding and pore-forming subunit ofanthrax toxin, will be used to specifically deliver the PA-bindingdomain of lethal factor (LFn) fused to the enzymatic domain ofdiphtheria toxin (DTA) or Ricin (Rcn) into ANTXR2+ cells in the joints,including nociceptor neurons, macrophages and osteoclasts. These toxinswill be injected into osteoarthritic or rheumatoid arthritic joints tosilence pain and joint destruction.

In mouse models, we will deliver locally subcutaneously or into thejoints the above anthrax toxin molecules or molecular combinations totest the efficacy of pain blockade or joint preservation in animalmodels of: osteoarthritis (e.g., via monoiodoacetate injection), muscleinjury (e.g., cardiotoxin induced damage), rheumatoid arthritis (e.g.,K/BxN Serum transfer arthritis), chemotherapy induced neuropathy (e.g.,Paclitaxel), and cancer pain (e.g., Ehrlich cell model)

Pain behavioral testing will assay the effects on mechanical and/orthermal hyperalgesia. We will also perform electrophysiology on primarynociceptor neurons to detect delivery of intracellular toxins intoneurons and neuronal activity block. Joint pathology will be analyzed bymeasures of inflammation and histological analysis.

Of note, we can also utilize this Anthrax Toxin platform to deliver theenzymatic moiety of Botulinum Toxin (BTx).

FIGS. 1A and 1B depict the specific and high level expression of thereceptor for the major anthrax toxin receptor within the dorsal rootganglia compared to 11 other nervous tissue types.

FIG. 2 compares whole transcriptome data between purified mousepain-sensing nociceptor neurons (Nav1.8-Cre/TdTomato+) vs. anothersomatosensory neuron subtype, proprioceptors (Parv-Cre/TdTomato+).Though they are related cell-types, pain-sensing Nav1.8+ nociceptorneurons show highly enriched expression of Antxr2 compared toproprioceptors. Antxr2 is >5-fold enriched in nociceptors vs.proprioceptor neurons (P-value<10⁻⁵).

Taken together, these data from FIGS. 1 and 2 show that Antxr2, thereceptor for anthrax toxin is highly enriched in nociceptor neuronscompared to other CNS tissues and to proprioceptor neurons. Thisstrongly indicates that Antxr2 can be utilized to target pain-sensingneurons specifically versus other neuronal subtypes.

Example 2 Assessment of In Vitro Efficacy (1)

We experimented to determine whether anthrax toxin components can bedelivered to nociceptors in culture. This would be testing for theinhibition of protein synthesis in a mammalian cell in presence of thecombination PA and the engineered fusion protein LFn-DTA, testing forthe increase in intracellular cAMP levels in a mammalian cell inpresence of the combination of PA and EF, and testing for the inhibitionof MAPK signaling in a mammalian cell in presence of the combination PAand LF.

The A chain of diphtheria toxin (DTA) is used as the intracellularenzymatic toxin for proof of principle that the anthrax delivery systemcan be use to deliver toxins into neurons. DTA catalyzes theADP-ribosylation of EF-2 and inhibits protein synthesis. The fusionprotein, LFn-DTA, is frequently used to assay for PA-mediatedtranslocation into the cytoplasm. Shown previously in Milne et al. Mol.Microbiol. Feb; 15(4):661-6, (1995) and in Liao et al. Chem. Bio. Chem.,15(16): 2458-2466, (2014), CHO-K1 cells treated with 20 nM PA togetherwith the fusion protein, LFn-DTA or DTA-LFn, completely stopped proteinsynthesis at the femtomolar or picomolar concentration of a fusionproteins.

For our experiment, DRG neurons were harvested from wild-type B6 miceand cultured overnight at a cell density of ˜2000 neurons/well. Cellswere treated with varying concentrations of PA and LFn-DTA for 6 hrs at37° C. in the presence of 3H-leu (400 nM). Protein synthesis wasmeasured using radiolabeled leucine (3H-leu), newly synthesized proteinwould incorporate 3H-leu into the new protein. After the incubationperiod, the neurons were wash with F12K medium (leu-free), thenincubated with 3H-leu in F12K medium (leu-free), followed by washingwith PBS, and then finally lysed in scintillation fluid for themeasurement of the incorporated 3H-leu radioactivity in newlysynthesized protein. Control experiments were performed with PA but withno LFn-DTA added. Background 3H-leu radioactivity was subtracted fromthe experimental radioactivity, and the subtracted data was normalizedto that of untreated neurons. No significant inhibition of proteinsynthesis was observed when the neurons were treated with PA alone(FIGS. 3 and 4). Therefore, PA alone does not affect protein synthesisin neurons. However, PA and LFn-DTA potently inhibits protein synthesisat nanomolar concentrations in cultured neurons. (FIGS. 5 and 6).

Sequence listings PA, NCBI Ref Seq: NP_052806 SEQ ID NO: 1,    1MKKRKVLIPL MALSTILVSS TGNLEVIQAE VKQENRLLNE SESSSQGLLG YYFSDLNFQA   61PMVVTSSTTG DLSIPSSELE NIPSENQYFQ SAIWSGFIKV KKSDEYTFAT SADNHVTMWV  121DDQEVINKAS NSNKIRLEKG RLYQIKIQYQ RENPTEKGLD FKLYWTDSQN KKEVISSDNL  181QLPELKQKSS NSRKKRSTSA GPTVPDRDND GIPDSLEVEG YTVDVKNKRT FLSPWISNIH  241EKKGLTKYKS SPEKWSTASD PYSDFEKVTG RIDKNVSPEA RHPLVAAYPI VHVDMENIIL  301SKNEDQSTQN TDSQTRTISK NTSTSRTHTS EVHGNAEVHA SFFDIGGSVS AGFSNSNSST  361VAIDHSLSLA GERTWAETMG LNTADTARLN ANIRYVNTGT APIYNVLPTT SLVLGKNQTL  421ATIKAKENQL SQILAPNNYY PSKNLAPIAL NAQDDFSSTP ITMNYNQFLE LEKTKQLRLD  481TDQVYGNIAT YNFENGRVRV DTGSNWSEVL PQIQETTARI IFNGKDLNLV ERRIAAVNPS  541DPLETTKPDM TLKEALKIAF GFNEPNGNLQ YQGKDITEFD FNFDQQTSQN IKNQLAELNA  601TNIYTVLDKI KLNAKMNILI RDKRFHYDRN NIAVGADESV VKEAHREVIN SSTEGLLLNI  661DKDIRKILSG YIVEIEDTEG LKEVINDRYD MLNISSLRQD GKTFIDFKKY NDKLPLYISN  721PNYKVNVYAV TKENTIINPS ENGDISINGI KKILIFSKKG YEIGDiptheria toxin NCBI Ref Seq: WP_003850266 SEQ ID NO: 2    1MSRKLFASIL IGALLGIGAP PSAHAGADDV VDSSKSFVME NFSSYHGTKP GYVDSIQKGI   61QKPKSGTQGN YDDDWKGFYS TDNKYDAAGY SVDNENPLSG KAGGVVKVTY PGLTKVLALK  121VDNAETIKKE LGLSLTEPLM EQVGTEEFIK RFGDGASRVV LSLPFAEGSS SVEYINNWEQ  181AKALSVELEI NFETRGKRGQ DAMYEYMAQA CAGNRVRRSV GSSLSCINLD WDVIRDKTKT  241KIESLKEHGP IKNKMSESPN KTVSEEKAKQ YLEEFHQTAL EHPELSELKT VTGTNPVFAG  301ANYAAWAVNV AQVIDSETAD NLEKTTAALS ILPGIGSVMG IADGAVHHNT EEIVAQSIAL  361SSLMVAQAIP LVGELVDIGF AAYNFVESII NLFQVVHNSY NRPAYSPGHK TQPFLHDGYA  421VSWNTVEDSI IRTGFQGESG HDIKITAENT PLPIAGVLLP TIPGKLDVNK SKTHISVNGR  481KIRMRCRAID GDVTFCRPKS PVYVGNGVHA NLHVAFHRSS SEKIHSNEIS SDSIGVLGYQ  541KTVDHTKVNS KLSLFFEIKSPseudomonas aeruginosa exotoxin A (PTx) NCBI Ref Seq: NP_249839SEQ ID NO: 3    1MHLTPHWIPL VASLGLLAGG SFASAAEEAF DLWNECAKAC VLDLKDGVRS SRMSVDPAIA   61DTNGQGVLHY SMVLEGGNDA LKLAIDNALS ITSDGLTIRL EGGVEPNKPV RYSYTRQARG  121SWSLNWLVPI GHEKPSNIKV FIHELNAGNQ LSHMSPIYTI EMGDELLAKL ARDATFFVRA  181HESNEMQPTL AISHAGVSVV MAQAQPRREK RWSEWASGKV LCLLDPLDGV YNYLAQQRCN  241LDDTWEGKIY RVLAGNPAKH DLDIKPTVIS HRLHFPEGGS LAALTAHQAC HLPLETFTRH  301RQPRGWEQLE QCGYPVQRLV ALYLAARLSW NQVDQVIRNA LASPGSGGDL GEAIREQPEQ  361ARLALTLAAA ESERFVRQGT GNDEAGAASA DVVSLTCPVA AGECAGPADS GDALLERNYP  421TGAEFLGDGG DISFSTRGTQ NWTVERLLQA HRQLEERGYV FVGYHGTFLE AAQSIVFGGV  481RARSQDLDAI WRGFYIAGDP ALAYGYAQDQ EPDARGRIRN GALLRVYVPR SSLPGFYRTG  541LTLAAPEAAG EVERLIGHPL PLRLDAITGP EEEGGRLETI LGWPLAERTV VIPSAIPTDP  601RNVGGDLDPS SIPDKEQAIS ALPDYASQPG KPPREDLKBotulinum toxin NCBI Ref Seq: YP_001386738 SEQ ID NO: 4    1MPFVNKQFNY KDPVNGVDIA YIKIPNAGQM QPVKAFKIHN KIWVIPERDT FTNPEEGDLN   61PPPEAKQVPV SYYDSTYLST DNEKDNYLKG VTKLFERIYS TDLGRMLLTS IVRGIPFWGG  121STIDTELKVI DTNCINVIQP DGSYRSEELN LVIIGPSADI IQFECKSFGH EVLNLTRNGY  181GSTQYIRFSP DFTFGFEESL EVDTNPLLGA GKFATDPAVT LAHELIHAGH RLYGIAINPN  241RVFKVNTNAY YEMSGLEVSF EELRTFGGHD AKFIDSLQEN EFRLYYYNKF KDIASTLNKA  301KSIVGTTASL QYMKNVFKEK YLLSEDTSGK FSVDKLKFDK LYKMLTEIYT EDNFVKFFKV  361LNRKTYLNFD KAVFKINIVP KVNYTIYDGF NLRNTNLAAN FNGQNTEINN MNFTKLKNFT  421GLFEFYKLLC VRGIITSKTK SLDKGYNKAL NDLCIKVNNW DLFFSPSEDN FTNDLNKGEE  481ITSDTNIEAA EENISLDLIQ QYYLTFNFDN EPENISIENL SSDIIGQLEL MPNIERFPNG  541KKYELDKYTM FHYLRAQEFE HGKSRIALTN SVNEALLNPS RVYTFFSSDY VKKVNKATEA  601AMFLGWVEQL VYDFTDETSE VSTTDKIADI TIIIPYIGPA LNIGNMLYKD DFVGALIFSG  661AVILLEFIPE IAIPVLGTFA LVSYIANKVL TVQTIDNALS KRNEKWDEVY KYIVTNWLAK  721VNTQIDLIRK KMKEALENQA EATKAIINYQ YNQYTEEEKN NINFNIDDLS SKLNESINKA  781MININKFLNQ CSVSYLMNSM IPYGVKRLED FDASLKDALL KYIYDNRGTL IGQVDRLKDK  841VNNTLSTDIP FQLSKYVDNQ RLLSTFTEYI KNIINTSILN LRYESNHLID LSRYASKINI  901GSKVNFDPID KNQIQLFNLE SSKIEVILKN AIVYNSMYEN FSTSFWIRIP KYFNSISLNN  961EYTIINCMEN NSGWKVSLNY GEIIWTLQDT QEIKQRVVFK YSQMINISDY INRWIFVTIT 1021NNRLNNSKIY INGRLIDQKP ISNLGNIHAS NNIMFKLDGC RDTHRYIWIK YFNLFDKELN 1081EKEIKDLYDN QSNSGILKDF WGDYLQYDKP YYMLNLYDPN KYVDVNNVGI RGYMYLKGPR 1141GSVMTTNIYL NSSLYRGTKF IIKKYASGNK DNIVRNNDRV YINVVVKNKE YRLATNASQA 1201GVEKILSALE IPDVGNLSQV VVMKSKNDQG ITNKCKMNLQ DNNGNDIGFI GFHQFNNIAK 1261LVASNWYNRQ IERSSRTLGC SWEFIPVDDG WGERPLTetanus toxin NCBI Ref Seq: WP_023439719 SEQ ID NO: 5    1MPITINNFRY SDPVNNDTII MMEPPYCKGL DIYYKAFKIT DRIWIVPERY EFGTKPEDFN   61PPSSLIEGAS EYYDPNYLRT DSDKDRFLQT MVKLFNRIKN NVAGEALLDK IINAIPYLGN  121SYSLLDKFDT NSNSVSFNLS EQDPSGATTK SAMLTNLIIF GPGPVLNKNE VRGIVLRVDN  181KNYFPCRDGF GSIMQMAFCP EYIPTFDNVI ENITSLTIGK SKYFQDPALL LMHELIHVLH  241GLYGMQVSSH EIIPSKQEIY MQHTYPISAE ELFTFGGQDA NLISIDIKND LYEKTLNDYK  301AIANKLSQVT SCNDPNIDID SYKQIYQQKY QFDKDSNGQY IVNEDKFQIL YNSIMYGFTE  361IELGKKFNIK TRLSYFSMNH DPVKIPNLLD DTIYNDTEGF NIESKDLKSE YKGQNMRVNT  421NAFRNVDGSG LVSKLIGLCK KIIPPTNIRE NLYNRTASLT DLGGELCIKI KNEDLTFIAE  481KNSFSEEPFQ DETVSYNTKN KPLNFNYSLD KIILDYNLQS KITLPNDRTT PVTKGIPYAP  541KYKSNAASTI EIHNIDDNTI YQYLYAQKSP TTLQRITMTN SVDDALINST KIYSYFPSVI  601SKVNQGAQGI LFLQWVRDII DDFTNESSQK TTIDKISDVS TIVPYIGPAL NIVKQGYEGN  661FIGALETTGV VLLLEYIPEI TLPVIAALSI AESSTQKEKI IKTIDNFLEK RYEKWIEVYK  721LIKAKWLGTV NTQFQKRSYQ MYRSLEYQVD AIKKIIDYEY KIYSGPDKEQ IADEINNLKN  781KLEEKANKAM ININIFMRES SRSFLVNQMI NEAKKQLLEF DTQSKNILMQ YIKANSKFIG  841ITELKKLESK INKVFSTPIP FSYSKNLDCW VDNEEDIDVI LKKSTILNLD INNDIISDIS  901GFNSSVITYP DAQLVPGING KAIHLVNNES SEVIVHKAMD IEYNDMFNNF TVSFWLRVPK  961VSASHLEQYG TNEYSIISSM KKYSLSIGSG WSVSLKGNNL IWTLKDSAGE VRQITFSDLS 1021DKFNAYLANK WVFITITNDR LSSANLYING VLMKNAEITG LGAIREDNNI TLKLDRCNNN 1081NQYVSIDKFR IFCKALNPKE IEKLYTSYLS ITFLRDFWGN PLRYDTEYYL IPVASSSKDV 1141QLKNITDYMY LTNAPSYTNG KLNIYYRRLY SGLKFIIKRY TPNNEIDSFV KSGDFIKLYV 1201SYNNNEHIVG YPKDGNAFNN LDRILRVGYN APGIPLYKKM EAVKLRDLKT YSVQLKLYDD 1261KNASLGLVGI RNGQIGNDPN RDILIASNWY FNHLKDKTLT CDWYFVPTDE GWTNDEdema Factor NCBI Ref Seq: NP_052818 SEQ ID NO: 6    1MTRNKFIPNK FSIISFSVLL FAISSSQAIE VNAMNEHYTE SDIKRNHKTE KNKTEKEKFK   61DSINNLVKTE FTNETLDKIQ QTQDLLKKIP KDVLEIYSEL GGEIYFTDID LVEHKELQDL  121SEEEKNSMNS RGEKVPFASR FVFEKKRETP KLIINIKDYA INSEQSKEVY YEIGKGISLD  181IISKDKSLDP EFLNLIKSLS DDSDSSDLLF SQKFKEKLEL NNKSIDINFI KENLTEFQHA  241FSLAFSYYFA PDHRTVLELY APDMFEYMNK LEKGGFEKIS ESLKKEGVEK DRIDVLKGEK  301ALKASGLVPE HADAFKKIAR ELNTYILFRP VNKLATNLIK SGVATKGLNV HGKSSDWGPV  361AGYIPFDQDL SKKHGQQLAV EKGNLENKKS ITEHEGEIGK IPLKLDHLRI EELKENGIIL  421KGKKEIDNGK KYYLLESNNQ VYEFRISDEN NEVQYKTKEG KITVLGEKFN WRNIEVMAKN  481VEGVLKPLTA DYDLFALAPS LTEIKKQIPQ KEWDKVVNTP NSLEKQKGVT NLLIKYGIER  541KPDSTKGTLS NWQKQMLDRL NEAVKYTGYT GGDVVNHGTE QDNEEFPEKD NEIFIINPEG  601EFILTKNWEM TGRFIEKNIT GKDYLYYFNR SYNKIAPGNK AYIEWTDPIT KAKINTIPTS  661AEFIKNLSSI RRSSNVGVYK DSGDKDEFAK KESVKKIAGY LSDYYNSANH IFSQEKKRKI  721SIFRGIQAYN EIENVLKSKQ IAPEYKNYFQ YLKERITNQV QLLLTHQKSN IEFKLLYKQL  781NFTENETDNF EVFQKIIDEK Lethal Factor NCBI Ref Seq: NP_052803 SEQ ID NO: 7   1 MNIKKEFIKV ISMSCLVTAI TLSGPVFIPL VQGAGGHGDV GMHVKEKEKN KDENKRKDEE  61 RNKTQEEHLK EIMKHIVKIE VKGEEAVKKE AAEKLLEKVP SDVLEMYKAI GGKIYIVDGD 121 ITKHISLEAL SEDKKKIKDI YGKDALLHEH YVYAKEGYEP VLVIQSSEDY VENTEKALNV 181 YYEIGKILSR DILSKINQPY QKFLDVLNTI KNASDSDGQD LLFTNQLKEH PTDFSVEFLE 241 QNSNEVQEVF AKAFAYYIEP QHRDVLQLYA PEAFNYMDKF NEQEINLSLE ELKDQRMLSR 301 YEKWEKIKQH YQHWSDSLSE EGRGLLKKLQ IPIEPKKDDI IHSLSQEEKE LLKRIQIDSS 361 DFLSTEEKEF LKKLQIDIRD SLSEEEKELL NRIQVDSSNP LSEKEKEFLK KLKLDIQPYD 421 INQRLQDTGG LIDSPSINLD VRKQYKRDIQ NIDALLHQSI GSTLYNKIYL YENMNINNLT 481 ATLGADLVDS TDNTKINRGI FNEFKKNFKY SISSNYMIVD INERPALDNE RLKWRIQLSP 541 DTRAGYLENG KLILQRNIGL EIKDVQIIKQ SEKEYIRIDA KVVPKSKIDT KIQEAQLNIN 601 QEWNKALGLP KYTKLITFNV HNRYASNIVE SAYLILNEWK NNIQSDLIKK VTNYLVDGNG 661 RFVFTDITLP NIAEQYTHQD EIYEQVHSKG LYVPESRSIL LHGPSKGVEL RNDSEGFIHE 721 FGHAVDDYAG YLLDKNQSDL VTNSKKFIDI FKEEGSNLTS YGRTNEAEFF AEAFRLMHST 781 DHAERLKVQK NAPKTFQFIN DQIKFIINSω-conotoxin M VII A NCBI Ref Seq: ADB93081 SEQ ID NO: 8    1MKLTCVVIVA VLLLTACQLI TADDSRGTQK HRALRSTTKL SMSTRCKGKG AKCSRLMYDC   61CTGSCRSGKC G μ-conotoxin Swiss-Prot: P15472.1 SEQ ID NO: 9    1ACSGRGSRCP PQCCMGLRCG RGNPQKCIGA HEDV δ-conotoxin NCBI ID: AKD43185SEQ ID NO: 10    1 LNKRCAGIGS FCGLPGLVDC CSGRCFIVCL P Shiga toxin A-partSEQ ID NO: 11,KEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSGTGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMASDEFPSMCPADGRVRGITHNKILWDSSTLGAILMRRTISSShiga toxin B-part: SEQ ID NO: 12,TPDCVTGKVEYTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITGMTVTIKTNACHNGGGFSEVIFRExotoxin A, A-part and B-part SEQ ID NO: 13AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTSLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK Cholera toxin Cholera toxin, A1 component SEQ ID NO: 14,NDDKLYRADSRPPDEIKQSGGLMPRGQSEYFDRGTQMNINLYDHARGTQTGFVRHDDGYVSTSISLRSAHLVGQTILSGHSTYYLYVLATAPNMFNVNDVLGAYSPHPDEQEVSALGGIPYSQIYGWYRVHFGVLDEQLHRNRGYRDRYYSNLDIAPAADGYGLAGFPPEHRAWREEPWIHHAPPGCGNAPRSSCholera toxin, A2 component SEQ ID NO: 15,MSNTCDEKTQSLGVKFLDEYQSKVKRQIFSGYQSDIDTHNRIKDELCholera toxin, B component SEQ ID NO: 16,TPQNITDLCAEYHNTQIYTLNDKIFSYTESLAGKREMAIITFKNGAIFQVEVPSSQHIDSQKKAIERMKDTLRIAYLTEA KVEKLCTWNNKTPHAIAAISMANCatalytic chain of tetanus (the L-chain sequence) SEQ ID NO: 17,MPITINNFRYSDPVNNDTIIMMEPPYCKGLDIYYKAFKITDRIWIVPERYEFGTKPEDFNPPSSLIEGASEYYDPNYLRTDSDKDRFLQTMVKLFNRIKNNVAGEALLDKIINAIPYLGNSYSLLDKFDTNSNSVSFNLLEQDPSGATTKSAMLTNLIIFGPGPVLNKNEVRGIVLRVDNKNYFPCRDGFGSIMQMAFCPEYVPTFDNVIENITSLTIGKSKYFQDPALLLMHELIHVLHGLYGMQVSSHEIIPSKQEIYMQHTYPISAEELFTFGGQDANLISIDIKNDLYEKTLNDYKAIANKLSQVTSCNDPNIDIDSYKQIYQQKYQFDKDSNGQYIVNEDKFQILYNSIMYGFTEIELGKKFNIKTRLSYFSMNHDPVKIPNLLDDTIYNDTEGFNIESKDLKSEYKGQNMRVNTNAFRNVDGSGLVSKLIGLCKKIIPPTNIRENLYNRT Ricin toxin Ricin toxin, A component SEQ ID NO: 18,IFPKQYPIINFTTAGATVQSYTNFIRAVRGRLTTGADVRHEIPVLPNRVGLPINQRFILVELSNHAELSVTLALDVTNAYVVGYRAGNSAYFFHPDNQEDAEAITHLFTDVQNRYTFAFGGNYDRLEQLAGNLRENIELGNGPLEEAISALYYYSTGGTQLPTLARSFIICIQMISEAARFQYIEGEMRTRIRYNRRSAPDPSVITLENSWGRLSTAIQESNQGAFASPIQLQRRNGSKFSVYDVSILIPIIALMVYRCAPPPSSQF Ricin toxin , B componentSEQ ID NO: 19,ADVCMDPEPIVRIVGRNGLCVDVRDGRFHNGNAIQLWPCKSNTDANQLWTLKRDNTIRSNGKCLTTYGYSPGVYVMIYDCNTAATDATRWQIWDNGTIINPRSSLVLAATSGNSGTTLTVQTNIYAVSQGWLPTNNTQPFVTTIVGLYGLCLQANSGQVWIEDCSSEKAEQQWALYADGSIRPQQNRDNCLTSDSNIRETVVKILSCGPASSGQRWMFKNDGTILNLYSGLVLDVRASDPSLKQIILYPLHGDPNQIWLPLFBTx-A light chain (native): BTx-A Amino acids 1-448 SEQ ID NO: 20,MPFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKEDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKBTx-A light chain Amino acids 1-430 SEQ ID NO: 21,MPFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCBTx-B light chain (native): BTx-B Amino acids 1-441 SEQ ID NO: 22,MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFNKSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLGDRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNHFASREGEGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETNIAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKAINKQAYEEISKEHLAVYKIQMCKSVKBTx-B Amino acids 1-437 SEQ ID NO: 23,MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFNKSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLGDRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNHFASREGFGGIMQMKFCPEYVSVENNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLYGIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETNIAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKAINKQAYEEISKEHLAVYKIQMCBTx-C1 light chain (native): BoN/T-C1 Amino acids 1-449 SEQ ID NO: 24,MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVTSPKSGYYDPNYLSTDSDKDPFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDVDFNSVDVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTFAAQEGFGALSIISISPRFMLTYSNATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQYNVKLEYAEIYAFGGPTIDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKLIRKYRFVVESSGEVTVNRNKFVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNVLFMGQNLSRNPALRKVNPENMLYLFTKFCHKAIDGRSLYNKBTx-D light chain (native): BoN/T-D Amino acids 1-442 SEQ ID NO: 25,MTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSKPPRPTSKYQSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDTFDFTRHTTNIAVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLTFSDVTSNQSSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYTFGGLDVEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNFVVNIDKFNSLYSDLTNVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNIENSGQNIERNPALQKLSSESVVDLFTKVCLRLTKBTx-E light chain (native): BTx-E Amino acids 1-422 SEQ ID NO: 26,MPTINSFNYNDPVNNRTILYIKPGGCQQFYKSFNIMKNIWIIPERNVIGTIPQDFLPPTSLKNGDSSYYDPNYLQSDQEKDKFLKIVTKIFNRINDNLSGRILLEELSKANPYLGNDNTPDGDFIINDASAVPIQFSNGSQSILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHGFGSIAIVTFSPEYSFRFKDNSMNEFIQDPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPLITNIRGTNIEEFLTEGGTDLNIITSAQSNDIYTNLLADYKKIASKLSKVQVSNPLLNPYKDVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLATKFQVKCRQTYIGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITGRGLVKKIIRFCKNIVSVKGIRBTx-F light chain (native): BTx-F Amino acids 1-436 SEQ ID NO: 27,MPVAINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTNPSDFDPPASLKNGSSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGKVLLQEISYAKPYLGNDHIPIDEFSPVTRITSVNIKLSTNVESSMLLNLLVLGAGPDIFESCCYPVRKLIDPDVVYDPSNYGFGSINIVTFSPEYEYTFNDISGGHNSSTESFIADPAISLAHELIHALHGLYGARGVIYEETIEVKQAPLMIAEKPIRLEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATRLSEVNSAPPEYDINEYKDYFQWKYGLDKNADGSYTVNENKFNEIYKKLYSFTESDLANKFKVKCRNTYFIKYEFLKVPNLLDDDIYTVSEGFNIGNLAVNNRGQSIKLNPKIIDSIPDKGLVEKIVKFCKSVIPRKBTx-G light chain (native): BTx-G Amino acids 1-442 SEQ ID NO: 28,MPVNIKKFNYNDPINNDDIIMMEPFNDPGPGTYYKAFRIIDRIWIVPERFTYGFQPDQFNASTGVFSKDVYEYYDPTYLKTDAEKDKFLKTMIKLFNRINSKPSGQRLLDMIVDAIPYLGNASTPPDKFAANVANVSINKKIIQPGAEDQIKGLMTNLIIFGPGPVLSDNFTDSMIMNGHSPISEGFGARMMIRFCPSCLNVFNNVQENKDTSIFSRRAYFADPALTLMHELIHVLHGLYGIKISNLPITPNTKEFFMQHSDPVQAEELYTFGGHDPSVISPSTDMNIYNKALQNFQDIANRLNIVSSAQGSGIDISLYKQIYKNKYDFVEDPNGKYSVDKDKFDKLYKALMFGFTETNLAGEYGIKTRYSYFSEYLPPIKTEKLLDNTIYTQNEGFNIASKNLKTEFNGQNKAVNKEAYEEISLEHLVIYRIAMCKPVMYKBTx-A (light chain and heavy chain translocation domain) Amino acids 1-872SEQ ID NO: 29,MPFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNBTx-A, (light chain and heavy chain translocation domain) Amino acids 1-842SEQ ID NO: 30,MPFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNBTx-B (light chain and heavy chain translocation domain) Amino acids 1-863SEQ ID NO: 31,MPVTINNFNY NDPIDNNNII MMEPPFARGT GRYYKAFKIT DRIWIIPERY TFGYKPEDFN KSSGIFNRDVCEYYDPDYLN TNDKKNIFLQ TMIKLFNRIK SKPLGEKLLE MIINGIPYLG DRRVPLEEFN TNIASVTVNKLISNPGEVER KKGIFANLII FGPGPVLNEN ETIDIGIQNH FASREGFGGI MQMKFCPEYV SVFNNVQENKGASIFNRRGY FSDPALILMH ELIHVLHGLY GIKVDDLPIV PNEKKFFMQS TDAIQAEELY TFGGQDPSIITPSTDKSIYD KVLQNFRGIV DRLNKVLVCI SDPNININIY KNKFKDKYKF VEDSEGKYSI DVESFDKLYKSLMFGFTETN IAENYKIKTR ASYFSDSLPP VKIKNLLDNE IYTIEEGFNI SDKDMEKEYR GQNKAINKQAYEEISKEHLA VYKIQMCKSV KAPGICIDVD NEDLFFIADK NSFSDDLSKN ERIEYNTQSN YIENDFPINELILDTDLISK IELPSENTES LTDFNVDVPV YEKQPAIKKI FTDENTIFQY LYSQTFPLDI RDISLTSSFDDALLFSNKVY SFFSMDYIKT ANKVVEAGLF AGWVKQIVND FVIEANKSNT MDKIADISLI VPYIGLALNVGNETAKGNFE NAFEIAGASI LLEFIPELLI PVVGAFLLES YIDNKNKIIK TIDNALTKRN EKWSDMYGLIVAQWLSTVNT QFYTIKEGMY KALNYQAQAL EEIIKYRYNI YSEKEKSNIN IDFNDINSKL NEGINQAIDNINNFINGCSV SYLMKKMIPL AVEKLLDFDN TLKKNLLNYI DENKLYLIGS AEYEKSKVNK YLKTIMPFDLSIYTNDTILI EMFNKYNSEI LNN

1. A fusion protein comprising: a) a botulinum neurotoxin (BTx) or a tetanus neurotoxin (TTx), and b) an anthrax toxin protective antigen (PA), or a C-terminal receptor-binding domain of PA, wherein part (a) and (b) are linked or fused together.
 2. The fusion protein of claim 1, wherein the BTx or TTx comprises a BTx or TTx enzymatic moiety and the translocation signal.
 3. The fusion protein of claim 2, wherein the BTx enzymatic moiety or translocation signal is selected from the BTx light chain and heavy chain domains of any one of BTx/A, BTx/B, BTx/C, BTx/D, BTx/E, BTx/F, BTx/G, and a non-Clostridial botulinum-like toxin.
 4. The fusion protein of claim 2, wherein the BTx or TTx enzymatic moiety or translocation signal is selected from the respective LC enzymatic moiety or H_(N) translocation peptide found in Table
 1. 5. The fusion protein of claim 1, wherein the part (a) and (b), or the enzymatic moiety and translocation peptide are linked by a linker peptide.
 6. The fusion protein of claim 5, wherein the linker peptide is 1-20 amino acids long.
 7. The fusion protein of claim 6, wherein the linker peptide is stable in human serum for at least 1 minute.
 8. The fusion protein of claim 7, wherein the linker peptide comprises at least one amino acid that is Gly or Ser.
 9. The fusion protein of claim 6, wherein the linker peptide does not comprise Lys and/or Arg.
 10. The fusion protein of claim 1, wherein the C-terminal receptor-binding domain of PA comprises a PAd4 domain, a PAd2 and a PAd4 domain, or PA63.
 11. The fusion protein of claim 1, wherein the PA or the C-terminal receptor-binding domain of PA is resistant to cleavage by a protease.
 12. The fusion protein of claim 11, wherein the the PA or the C-terminal receptor-binding domain of PA is resistant to furin cleavage, or Lys C cleavage or both.
 13. A fusion protein comprising: a) a non-cytotoxic protease, which protease is capable of cleaving a SNARE protein in a nociceptor neuron; and b) a protein capable of binding to an anthrax toxin protective antigen (PA) or a fragment thereof, wherein the PA or fragment binds a receptor expressed on the nociceptor neuron.
 14. The fusion protein of claim 13, wherein the non-cytotoxic protease comprises a clostridial neurotoxin L-chain.
 15. The fusion protein of claim 14, wherein the clostridial neurotoxin is botulinum neurotoxin (BTx) or tetanus neurotoxin (TTx).
 16. The fusion protein of claim 14, wherein the clostridial neurotoxin L-chain is selected from Table
 1. 17. The fusion protein of claim 13, wherein the PA-binding receptor expressed on the nociceptor neuron is ANTXR2 (CMG2).
 18. The fusion protein of claim 13, wherein the protein capable of binding to PA is: i) an anthrax toxin lethal factor (LF); or ii) an anthrax toxin edema factor (EF).
 19. The fusion protein of claim 18, wherein PA binding domain of LF is the N-terminal domain of LF, (abbreviated as LFPABD or LFn).
 20. The fusion protein of claim 18, wherein PA binding domain of EF is the N-terminal domain of EF, (abbreviated as EFPABD or EFn).
 21. A fusion protein comprising: a) a disulfide-containing peptide toxin (these include the channel blocking toxins having a cysteine-knot motif), which is capable of blocking ion channels in a nociceptor neuron; and b) a targeting moiety (TM) that is capable of binding to a binding site on the nociceptor neuron, wherein the nociceptor neuron expresses said ion channels therein (e.g., sodium or calcium or both sodium and calcium);
 22. The fusion protein of claim 21, wherein the disulfide-containing peptide toxin comprises a cysteine knot motif.
 23. The fusion protein of claim 21, wherein the disulfide-containing peptide toxin is a conotoxin, an agatoxin, a delta-palutoxin, a huwentotoxin or a ProTx II toxin.
 24. The fusion protein according to claim 21, wherein the TM is selected from the group consisting of: i) an anthrax toxin protective antigen (PA); ii) a C-terminal receptor-binding domain of PA; iii) a nociceptor neuron-binding protein.
 25. The fusion protein according to claim 24, wherein the PA or C-terminal receptor-binding domain of PA interacts with and binds the ANTXR2 (CMG2) receptor expressed on the nociceptor neuron. 26-29. (canceled)
 30. The fusion protein of claim 24, wherein the nociceptor neuron-binding protein is an antibody.
 31. The fusion protein of claim 30, wherein the antibody specifically binds to nerve growth factor receptor, the ANTXR2 receptor, or an ion-channel protein present on nociceptor neurons.
 32. The fusion protein of claim 31, wherein the ion-channel protein is selected from Nav1.7, Nav1.8 or Nav1.9.
 33. A fusion protein comprising: a) a disulfide-containing peptide toxin (this are channel blocking toxin having a cysteine-knot motif), which is capable of blocking sodium or calcium or both sodium and calcium channels in a nociceptor neuron; and b) a protein capable of binding to an anthrax toxin protective antigen (PA) or a fragment thereof, wherein the fragment binds a receptor expressed on the nociceptor neuron. 34-37. (canceled)
 38. A fusion protein comprising: a) an AB toxin; b) an anthrax toxin protective antigen (PA) or a fragment thereof, wherein the PA or fragment binds a receptor expressed on the nociceptor neuron; and c) a Translocation domain (TL) that is capable of translocating the protease from within an endosome, across the endosomal membrane and into the cytosol of the nociceptor neuron.
 39. The fusion protein of claim 38, wherein the AB toxin is selected from a. Ricin toxin, b. Cholera toxin A-part and B-part; c. Pseudomonas aeruginosa Exotoxin A A-part and B-part; d. Shiga toxin A-part and B-part; and e. Diphtheria toxin A-part and B-part.
 40. (canceled)
 41. The fusion protein of claim 38, wherein the PA fragment is a C-terminal receptor-binding domain of PA.
 42. The fusion protein of claim 38, wherein the TL is i) a clostridial neurotoxin translocation domain; ii) a holotoxin; or iii) a variant form of the holotoxin that has the toxin receptor-binding function of the AB toxin negated. 43-49. (canceled)
 50. A method for treatment of pain, the method comprising administering to a subject in need thereof the fusion protein of claim
 1. 51. A method of treating pain comprising administering to a subject in need thereof, native mature anthrax toxin protective antigen (PA) and anthrax toxin edema factor (EF), anthrax toxin lethal factor (LF) or any combination thereof. 52-69. (canceled)
 70. A fusion protein comprising: a) a botulinum neurotoxin N-terminal enzymatic domain of a botulinum neurotoxin (BTx) moiety (L chain of BTx), and b) a N-terminal domain (LFn) of anthrax toxin lethal factor, which domain binds to oligomeric forms of PA63, the proteolytically activated form of anthrax PA; or the N-terminal domain (EFn) of anthrax toxin edema factor, which domain binds to oligomeric forms of PA63, the proteolytically activated form of anthrax PAthe enzymatic domain, wherein part (a) is linked N-terminally or C-terminally or both N-terminally and C-terminally to part (b).
 71. The fusion protein of claim 70 further comprising an amino acid sequence defining a belt corresponding to the N-terminal part of the BTx H_(N) domain which is located at C-terminal side of the BTx moiety, and the belt is linked to the L chain.
 72. The fusion protein of claim 71, wherein the residue corresponding to the L chain junction with the belt of the BTx H_(N) domain has been cleaved.
 73. The fusion protein of claim 71, wherein the Cys residues in the BTx L chain and the belt corresponding to the N-terminal part of the BTx H_(N) domain have been changed to Ala, Ser, or Thr.
 74. The fusion protein of claim 70, further comprising at least one D-amino acid at the N-terminus of the fusion protein.
 75. The fusion protein of claim 70, further comprising a linker peptide between the BTx L moiety and the LFn or EFn domain. 76-79. (canceled)
 80. A composition comprising claim
 70. 81. (canceled)
 82. The composition of claim 80 and a native anthrax toxin protective antigen (PA) protein. 83-85. (canceled)
 86. A fusion protein comprising: a) anthrax toxin protective antigen (PA); an anthrax toxin protective antigen C-terminal receptor binding domain (PAd4); or a nociceptor neuron-binding protein linked to, b) a disulfide-containing peptide toxin.
 87. The fusion protein of claim 86, wherein the disulfide-containing peptide toxin is an inhibitor cysteine knot toxin.
 88. (canceled)
 89. The fusion protein of claim 86, further comprising a linker peptide between the PA, PAd4 or nociceptor-binding protein and the inhibitor cysteine knot toxin. 90-104. (canceled)
 105. A fusion protein comprising a disulfide-containing peptide toxin operably linked N-terminally or C-terminally or both N-terminally and C-terminally, or chemically crosslinked at one or more sites to the N-terminal domain (LFn) of anthrax toxin lethal factor, which domain binds to oligomeric forms of PA63, the proteolytically activated form of anthrax PA; or the N-terminal domain (EFn) of anthrax toxin edema factor, which domain binds to oligomeric forms of PA63, the proteolytically activated form of anthrax PA.
 106. The fusion protein of claim 105, further comprising a linker peptide between the LFn and the toxin or the EFn and the toxin. 107-115. (canceled)
 116. A fusion protein comprising an AB toxin fused to a linker peptide operably linked to a C-terminal receptor-binding domain of anthrax toxin protective antigen (PAd4 domain), wherein the fusion protein further comprises a translocation domain, a holotoxin or a mutant form of the holotoxin that has the toxin receptor-binding function of the AB toxin negated. 117-121. (canceled)
 122. The fusion protein of claim 116, wherein 1-60 consecutive amino acids from the N-terminal side adjacent to the native PAd4 domain are further incorporated between the AB toxin and the PAd4. 123-131. (canceled)
 132. A fusion protein comprising an N-terminal enzymatic domain (Chain A) together with the translocation/pore-forming domain from a tetanus neurotoxin (TTx) operably linked to a C-terminal receptor-binding domain of anthrax toxin protective antigen (PAd4 domain).
 133. The fusion protein of claim 132 further comprising a linker peptide between the TTx moiety and the PAd4 domain. 134-138. (canceled)
 139. The fusion protein of claim 132, wherein 1-60 consecutive amino acids from the N-terminal side adjacent to the native PAd4 domain are further incorporated between the BTx moiety and the PAd4. 140-148. (canceled)
 149. A nucleic acid encoding the fusion protein of claim
 132. 150. A vector comprising the nucleic acid of claim
 149. 151. The vector of claim 150, wherein the vector is a plasmid, a bacteriophage, a phagmid, a cosmid, a viral particle, or viral vector.
 152. A cell comprising the nucleic acid of claim
 149. 153. A method of producing fusion protein comprising: a) culturing the cell of claim 152 in conditions such that the fusion protein is expressed; and b) recovering the fusion protein.
 154. The fusion protein produced by the method of claim
 153. 155. The fusion protein of claim 1, which is glycosylated. 156-164. (canceled)
 165. A use of fusion protein of claim 132 for the treatment of pain.
 166. A use of fusion protein of claim 132 for the manufacture of a medicament for the treatment of pain.
 167. A method for treatment of pain, the method comprising administering to a subject in need thereof the composition from claim
 80. 168. The method of claim 167, wherein the administering is performed by intrathecal infusion or intra-cerebroventricular infusion or by an epidural injection into the central nervous system, or by peripheral administration using intradermal injection, subcutaneous injection, intramuscular injection, intraneural injection, or intra-articular injection.
 169. The method of claim 168, wherein the pain is selected from diabetic neuropathic pain, cancer pain, fibromyalgia and other systemic pain disorders.
 170. A method for treatment of nerve, joint, skin, visceral, bladder, or muscle pain comprising administering peripherally by intradermal injection, subcutaneous injection, intramuscular injection, intraneural injection, or intra-articular injection to a subject in need thereof the composition from claim
 80. 171. A method for treatment of diabetic neuropathic pain, cancer pain, fibromyalgia or other systemic pain disorders comprising administering by epidural injection, intrathecal infusion or intra-cerebroventricular infusion into the central nervous system of a subject in need thereof the composition from claim
 80. 172. The method of claim 167, wherein the composition is administered separately before, simultaneously or after administering a composition comprising an anthrax protective antigen (PA) in a pharmaceutically acceptable carrier, excipient or diluent.
 173. (canceled)
 174. A method of manufacture of a pharmaceutical composition of claim 80 for the treatment of pain, wherein the pharmaceutical composition comprising fusion protein and a pharmaceutically acceptable carrier or excipient.
 175. A fusion protein comprising: a. a botulinum neurotoxin (BTx) moiety, wherein the BTx moiety comprises an enzymatic moiety and a translocation domain and b. a targeting moiety comprising a C-terminal receptor-binding domain of an anthrax toxin protective antigen (PA), wherein the C-terminal receptor-binding domain of PA comprises a PAd4 domain, wherein part (a) and (b) are linked or fused together.
 176. A nucleic acid encoding a fusion protein comprising: a. a botulinum neurotoxin (BTx) moiety that comprises an enzymatic moiety and a translocation domain; and b. a targeting moiety comprising a C-terminal receptor-binding domain of anthrax toxin protective antigen (PA), wherein the C-terminal receptor-binding domain of PA comprises a PAd4 domain, wherein part (a) and (b) are linked or fused together. 